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Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is part of BioLegend's, "Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets".

Proper citation: BioLegend, Inc. 2017. Activation and Intracellular Staining of Whole Blood: For the Detection of Intracellular Cytokines and Other Intracellular Targets. protocols.io https://dx.doi.org/10.17504/protocols.io.hukb6uw Copy   

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Authors: Roey Angel, Eva Petrova, Ana Lara
Group: SoWa RI Anaerobic and Molecular Microbiology (public)
Summary: The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.

Proper citation: Roey Angel, Eva Petrova, Ana Lara 2021. RNA-Stable Isotope Probing. protocols.io https://dx.doi.org/10.17504/protocols.io.brqwm5xe Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: This kit is designed for the sequential positive selection of CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). Human monocytes express both CD14 and CD4. When using only CD4 Nanobeads for positive selection of human CD4 T cells, monocytes could be isolated along with the T cells. If this monocyte fraction does not impact your application, there is no need to address it. However, not including this population may be required. Thus, the first step in this kit is the depletion of CD14+ cells using a combination of biotin anti-human CD14 and Streptavidin Nanobeads. The second step is the positive selection of the CD4 T cells using directly conjugated CD4 Nanobeads. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Human CD4 T Cell Selection Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7z8hp9w Copy   

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Authors: Masahiro Ichikawa, Tomoaki Akiyama, Yasushi Tsujimoto, Keisuke Anan, Tadashi Yamakawa, Yasuo Terauchi
Summary: 1.IntroductionMany patients with diabetic mellitus who use insulin develop changes in the subcutaneous adipose tissue at the injection site. These lesions are called lipohypertrophy (LH). The prevalence of LH was reported to be 30-50% or more (1). In a randomized crossover study, injections into sites of LH showed blunted insulin absorption and increased variability compared to injections into normal adipose tissues (2).Since sites of LH reduce the absorption of insulin, patients with LH often use higher total daily doses (TDD) of insulin and have worsened glucose control and higher hyperglycemic hemoglobin (HbA1c) levels (3-5). LH may increase the risk of adverse clinical outcomes and the cost of healthcare.Recently, the effect of injection technique (IT) education to avoid the injection into LH and improve glycemic control on patients with LH has been reported (1, 6).IT education includes stopping injections into LH, stopping needle reuse, and rotating injection sites. However, no meta-analysis has been performed. Thus, to assess the impact of IT education on clinical and metabolic parameters in adults with LH, we will summarize current evidence.2.Research questionP: Patients with type 1 diabetes or type 2 diabetes who are injecting insulin and have LH.I: Injection technique educationC: standard careO: change in TDD3.Method3.1 ProtocolWe used a systematic review protocol template(dx.doi.org/10.17504/protocols.io.biqrkdv6). We followed the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 for preparing this protocol(7). We will publish this protocol in protocols.io (https://www.protocols.io/).3.2 Inclusion criteria of the articles for the review3.2.1 Type of studiesWe will include individual, cluster, and cross-over randomized trials that assess the injection technique education in patients with type 1 or type 2 diabetes who are injecting insulin and have LH. We will not apply language or country restrictions. We will include all papers including published, unpublished articles, abstract of conference and letter.We will exclude observational studies and non randomized controlled trials. We will not exclude studies based on the observation period or publication year.3.2.2 Study participantsWe will includepatients with type 1 or type 2 diabetes who have been on insulin injections for at least 1 year, with LH, 18 years of age or older, any gender, no HbA1c restriction, and no education about LH within the past 6 months. The diagnosis of LH were made by a physician or nurse when it is clinically visible, palpable, or visible on ultrasound (8). We will accept any number of insulin injections per day, or size of needle or length for insulin injections..We will exclude pregnant women, patients who wish to become pregnant, lactating women and patients taking medications that may cause LH (anti-retroviral or corticosteroid therapy).3.2.3 InterventionIT education: Stop injecting into the LH site, stop reusing needles, and rotate injection sites. We will also accept other definitions of IT education. Any education period is acceptable.3.2.4 ControlStandard care (no intervention, usual care).3.3 Type of outcomes3.3.1 Primary outcomes1.Change in total daily doses of insulinDefinition: Change in total daily doses of insulin from baselinePeriod: Time frame is the longest follow-up period after 3 months2. Change in HbA1cDefinition: Change in HbA1c from baselinePeriod: Time frame is the longest follow-up period after 3 months3. HypoglycemiaDefinition: Hypoglycaemia was defined as the occurrence of one or more symptoms of hypoglycaemia (such as palpitations, tiredness, sweating, strong hunger, dizziness and tremor) and a confirmed blood glucose meter reading of ≤ 60 mg/dL (4). We will also accept hypoglycemia as the original investigators defined.Period: during follow up period3.3.2 Secondary outcomes1. Change in proportion of patients with cured LHDefinition: as original investigators definedPeriod: Time frame is the longest follow-up period after 3 months2. All adverse eventsDefinition: definition of adverse events are set by original authors.Period: during follow up period3.4 Search method3.4.1 Electronic searchWe will search the following databases:1. the Cochrane Central Register of Controlled Trials (CENTRAL);2. MEDLINE via PubMed;3. EMBASE via ProQuest Dialog;See Appendix 1, 2, and 3 for the search strategies.3.4.2 Other resourcesWe will also search the following databases for ongoing or unpublished trials:1. the World Health Organization International Clinical Trials Platform Search Portal (ICTRP);2. ClinicalTrials.gov;See Appendix 4, 5 for the search strategies.We will check the reference lists of studies, including international guidelines as well as the reference lists of eligible studies and articles citing eligible studies. We will ask the authors of original studies for unpublished or additional data.3.5 Data collection and analysis3.5.1 Selection of the studiesTwo independent reviewers will screen titles and abstracts, followed by the assessment of the eligibility based on the full texts. We will contact original authors if relevant data is missing. Disagreements between the two reviewers will be resolved by discussion, and if this fails, a third reviewer will act as an arbiter.3.5.2 Data extraction and managementTwo reviewers will perform independent data extraction of the included studies using standardized data collection form. The form will include the information on the first author’s name, year of study publication, country, sample size, proportion of male participants, mean age of participants, the number of participants with type 1 diabetes mellitus/type 2 diabetes mellitus, estimated diabetic duration, mean duration of insulin treatment, intervention details, detection methods of LH and the outcome measures above. Any disagreements will be resolved by discussion, and if this fails, a third reviewer will act as an arbiter. 3.6 Assessment of risk of bias in included studiesTwo reviewers will evaluate the risk of bias independently using the Risk of Bias 2 tool(9). The effect of interest is the intention-to-treat effect; the effect of assignment to the interventions at baseline, regardless of whether the interventions are received as intended. Disagreements between the two reviewers will be discussed, and if this fails, a third reviewer will be acting as an arbiter, if necessary.3.7 Measures of treatment effectsWe will pool the relative risk ratios and the 95% confidence intervals (CIs) for the following binary variables: Hypoglycemia, proportion of patients with cured LH and all adverse eventsWe will pool the mean differences and the 95% CIs for the following continuous variables: HbA1c, total daily doses of insulinWe will summarize adverse events based on the definition by the original article, but we will not perform meta-analysis..3.8 Unit of analysis issuesClustering at the level of the enrolled units in cluster randomised studies In dealing with cluster-RCTs, for dichotomous data, we will apply the design effect and calculate effective sample size and number of events using the intracluster correlation coefficient (ICC) among each unit and the average cluster size, as described in Chapter 16.3.5 of the Cochrane Handbook (10). If the ICC has not been reported, we will use the ICC of a similar study as a substitute. For continuous data, only the sample size will be reduced; means and standard deviation will remain unchanged (10).Randomised cross-over studiesWe will consider only data from the first period.Multiple comparisonsAll intervention groups that are relevant to this review will be included.3.9 Handling of missing dataWe will ask not-presented data to the original authors.3.9.1 Missing outcomesWe will perform the intention-to-treat (ITT) analysis for all dichotomous data as much as possible.For continuous data, we will not impute missing data based on the recommendation by Cochrane handbook (10). We will perform meta-analysis about the available data in the original study.3.9.2 Missing statisticsWhen original studies only report standard error or p-value, we will calculate the standard deviation based on the method by Altman (11). If we could not obtain these values by contacting authors, standard deviation will be calculated by confidence interval and t-value based on the method by Cochrane handbook (10), or validated method (11). Validity of these methods will be analyzed by sensitivity analysis.3.10 Assessment of heterogeneityWe will evaluate the statistical heterogeneity by visual inspection of the forest plots and calculating the I2statistic (I2 values of 0% to 40%: might not be important; 30% to 60%: may represent moderate heterogeneity; 50% to 90%: may represent substantial heterogeneity; 75% to 100%: considerable heterogeneity). When there is substantial heterogeneity (I2> 50%), we will assess the reason of the heterogeneity. Cochrane Chi2 test (Q-test) will be performed for I2 statistic, and P value less than 0.10 will be defined as statistically significant.3.11 Assessment of reporting biasWe will search the clinical trial registry system (ClinicalTrials.gov and ICTRP) and will perform extensive literature search for unpublished trials. We will assess the potential publication bias by visual inspection of the funnel plot. Egger’s test will be performed as well. We will not conduct the test when we find less than 10 trials or trials which have similar sample size. We will assess the potential publication bias by visual inspection of the funnel plot3.12 Meta-analysisMeta-analysis will be performed using Review Manager software (RevMan 5.4). We will use a random-effects model.3.13 Subgroup analysisTo elucidate the influence of effect modifiers on results, we will evaluate the subgroup analyses of the primary outcomes on the following factors when sufficient data are available.1. age (2. baseline HbA1c 3. gender4. IT education protocols with versus without initial insulin reduction3.14 Sensitivity analysisWe will undertake the following sensitivity analyses for the primary outcomes to assess whether the results of the review are robust to the decisions made during the review process. 1. Exclusion of studies using imputed statistics.2. Exclusion of studies with high overall risk of bias4. Summary of findings tableSummary of findings table will be made for the following outcome based on the Cochrane handbook (10).We will include grading to evaluate the quality of evidence based on the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach for each Summary of findings table (13).1. Change in total daily doses of insulin2. Change in HbA1c3. Hypoglycemia5. Conflict of InterestThe authors declare no conflicts of interests.Appendix 1: CENTRAL search strategy#1[mh Amyloidosis]#2 lipohypertrophy:ti,ab#3 "insulin-derived amyloidosis":ti,ab #4lipos:ti,ab#5 "subcutaneous induration":ti,ab#6"subcutaneous nodules":ti,ab#7 #1 OR #2 OR #3 OR #4 OR #5 OR #6#8 [mh "Injections, Subcutaneous"]#9 [mh "Patient Education as Topic"]#10injection*:ti,ab#11 education*:ti,ab#12 (reus*:ti,ab AND needle*:ti,ab)#13 #8 OR #9 OR #10 OR #11 OR #12#14 #7 AND #13Appendix 2: MEDLINE (via PubMed) search strategy#1 "Amyloidosis"[Mesh]#2 "lipohypertrophy"[tiab]#3 "insulin-derived amyloidosis"[tiab]#4 "lipos"[tiab]#5 "subcutaneous induration"[tiab]#6 "subcutaneous nodules"[tiab]#7 #1 OR #2 OR #3 OR #4 OR #5 OR #6#8 "Injections, Subcutaneous"[Mesh]#9 "Patient Education as Topic"[Mesh]#10 injection*[tiab] #11 education*[tiab] #12 reus*[tiab] AND needle*[tiab]#13 #8 OR #9 OR #10 OR #11 OR #12#14 #7 AND #13#15 randomized controlled trial[pt]#16 controlled clinical trial[pt]#17 randomized[tiab]#18 placebo[tiab]#19 drug therapy[sh]#20 randomly[tiab]#21 trial[tiab]#22 groups[tiab]#23 #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22#24 animals[mh] NOT humans[mh]#25 #23 NOT #24#26 #14 AND #25Appendix 3: EMBASE (via ProQuest Dialog) search strategy#1 emb(amyloidosis) OR ti(lipohypertrophy) OR ab(lipohypertrophy) OR ti(insulin-derived amyloidosis) OR ab(insulin-derived amyloidosis) OR ti(lipos) OR ab(lipos) OR ti(subcutaneous induration) OR ab(subcutaneous induration) OR ti(subcutaneous nodules) OR ab(subcutaneous nodules) #2 exact(subcutaneous drug administration) OR exact(patient education) OR ti(injection*) OR ab(injection*) OR ti(education*) OR ab(education*) OR (ti(reus*) OR ab(reus*)) AND (ti(needle*) OR ab(needle*)) #3 (ab(random*) OR ti(random*)) OR (ab(clinical NEAR/1 trial*) OR ti(clinical NEAR/1 trial*)) OR (EMB.EXACT("health care quality"))#4 #1 AND #2 AND #3Appendix 4: ICTRP search strategyParticipant: lipohypertrophy OR insulin-derived amyloidosis OR subcutaneous induration OR subcutaneous nodulesIntervention: injection OR education OR (reus AND needle)Appendix 5: ClinicalTrials.gov search strategyCondition or disease: lipohypertrophy OR insulin-derived amyloidosis OR subcutaneous induration OR subcutaneous nodulesIntervention: injection OR education OR (reus AND needle)References1.Chen L, Xing Q, Li J, Zhou J, Yuan Y, Wan Y, et al. Injection Technique Education in Patients with Diabetes Injecting Insulin into Areas of Lipohypertrophy: A Randomized Controlled Trial. Diabetes Ther. 2021.2.Famulla S, Hövelmann U, Fischer A, Coester H-V, Hermanski L, Kaltheuner M, et al. Insulin Injection Into Lipohypertrophic Tissue: Blunted and More Variable Insulin Absorption and Action and Impaired Postprandial Glucose Control. Diabetes Care. 2016;39(9):1486-92.3.Frid AH, Hirsch LJ, Menchior AR, Morel DR, Strauss KW. Worldwide Injection Technique Questionnaire Study: Injecting Complications and the Role of the Professional. Mayo Clinic proceedings. 2016;91(9):1224-30.4.Blanco M, Hernández MT, Strauss KW, Amaya M. Prevalence and risk factors of lipohypertrophy in insulin-injecting patients with diabetes. Diabetes Metab. 2013;39(5):445-53.5.Ji L, Sun Z, Li Q, Qin G, Wei Z, Liu J, et al. Lipohypertrophy in China: Prevalence, Risk Factors, Insulin Consumption, and Clinical Impact. Diabetes Technol Ther. 2017;19(1):61-7.6.Campinos C, Le Floch JP, Petit C, Penfornis A, Winiszewski P, Bordier L, et al. An Effective Intervention for Diabetic Lipohypertrophy: Results of a Randomized, Controlled, Prospective Multicenter Study in France. Diabetes Technol Ther. 2017;19(11):623-32.7. Shamseer L, Moher D, Clarke M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015;349(jan02 1):g7647-g7647. doi:10.1136/bmj.g76478.Deg N,Zhang X, Zhao F, Wang Y, He H. Prevalence of lipohypertrophy in insulin-treated diabetes patients: A systematic review and meta-analysis. 2018;9(3):536-43.9. Sterne JAC, Savović J, Page MJ, et al. RoB 2: a revised tool for assessing risk of bias in randomised trials. BMJ. August 2019:l4898. doi:10.1136/bmj.l489810. Higgins JPT, Thomas J, Chandler J, et al. Cochrane Handbook for Systematic Reviews of Interventions version 6.0 (updated July 2019). Cochrane, 2019.11. Furukawa T a, Barbui C, Cipriani A, Brambilla P, Watanabe N. Imputing missing standard deviations in meta-analyses can provide accurate results. J Clin Epidemiol. 2006;59(1):7-10. doi:10.1016/j.jclinepi.2005.06.00612.Duke S, Colagiuri S, Colagiuri R. Individual patient education for people with type 2 diabetes mellitus. Cochrane Database Syst Rev. 2009;21(1):CD00526813. Guyatt G, Oxman AD, Akl E a, et al. GRADE guidelines: 1. Introduction-GRADE evidence profiles and summary of findings tables. J Clin Epidemiol. 2011;64(4):383-394. doi:10.1016/j.jclinepi.2010.04.026

Proper citation: Masahiro Ichikawa, Tomoaki Akiyama, Yasushi Tsujimoto, Keisuke Anan, Tadashi Yamakawa, Yasuo Terauchi 2021. Efficacy of injection technique education in diabetes with lipohypertrophy: a systematic review and meta-analysis protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bteenjbe Copy   

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Authors: Jernej Turnsek, Chris Dupont
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Here we present an efficient way to grow Tp colonies inside a 0.25% superclean agar matrix. This protocol could possibly be applicable to other marine microeukaryotes that are problematic to grow on fully solid support.

Proper citation: Jernej Turnsek, Chris Dupont 2017. Pour plating of Thalassiosira pseudonana (Tp). protocols.io https://dx.doi.org/10.17504/protocols.io.i6qchdw Copy   

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Authors: Bryon Drown, Kelleher Research Group
Group: Kelleher Research Group
Summary: Methanol-chloroform-water (MCW) precipitation is a rapid method for removing interferring species from protein samples prior to LC-MS analysis. MCW precipitation can effectively remove both ionic and non-ionic detergents and salts. Precipitating less than 1 µg protein is challenging yet possible with careful pipetting.

Proper citation: Bryon Drown, Kelleher Research Group 2021. Methanol-Chloroform-Water Precipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.biknkcve Copy   

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Authors: Anika Wiegard
Group: Axmann Lab
Summary: This protocol can be used for:(i) heterologous expression of GST-tagged proteins from pGEX-6P1 based expression vectors in E. coli.(ii) purification of recombinant proteins via affinity chromatography using glutathione-agarose or glutathione-sepharose (GST tagged protein can be eluted with glutathione. Alternatively, the tag can be cleaved off by prescission protease)(iii) further purification of the eluted protein via anion exchange chromatographyThis protocol was modified fromWiegard A, Dörrich AK, Deinzer HT, Beck C, Wilde A, Holtzendorff J, Axmann IM: Biochemical analysis of three putative KaiC clock proteins from Synechocystis sp. PCC 6803 suggests their functional divergence. Microbiology 2013, 159, 948-958Snijder J, Schuller JM, Wiegard A, Lössel, P, Schmelling NM, Axmann IM, Plitzko JM, Förster F, Heck AJR: Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science 2017, 355(6330):1181-1184

Proper citation: Anika Wiegard 2018. Expression and purification of (GST-tagged) (Kai) proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.k68czhw Copy   

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Authors: Priota Islam
Group: Behavioural Genomics

Proper citation: Priota Islam 2019. Food preference assay of C Elegans. protocols.io https://dx.doi.org/10.17504/protocols.io.yb5fsq6 Copy   

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Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab

Proper citation: Maria Rubio-Brotons 2017. Transient transfection of unicellular relative to animals Corallochytrium limacisporum with electroporation using Neon SystemVersion 1. protocols.io https://dx.doi.org/10.17504/protocols.io.hmwb47e Copy   

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Authors: Reyka Jayasinghe, Li Ding, Feng Chen
Summary: Single-cell Isolation for solid tumors Ding Lab 2021

Proper citation: Reyka Jayasinghe, Li Ding, Feng Chen 2021. WU sc-prep Protocol for Solid Tumors v2.1. protocols.io https://dx.doi.org/10.17504/protocols.io.bsnqnddw Copy   

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Authors: Stephen Floor
Group: Stephen Floor Lab
Summary: This protocol is designed for 'around-the-horn' or 'divergent' PCR, where primers go around most or all of a plasmid but are pointed away from each other so they generate a linear product. Note that this protocol is written for Q5 polymerase, but works fine with other polymerases. To switch polymerases, just change the PCR reaction setup.

Proper citation: Stephen Floor 2018. Around-the-horn PCR and cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.rf2d3qe Copy   

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Authors: Cristina Moraru & Elke Allers
Group: VERVE Net, Sullivan Lab
Summary: This protocol combines phage gene detection with rRNA detection for the identification of host cells and detection of free phage particles.

Proper citation: Cristina Moraru & Elke Allers 2016. phageFISH Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.c9iz4d Copy   

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Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program
Summary: The goal of the Integrated Islet Distribution Program (IIDP) is to develop a uniform standardized shipping method among all the subcontracted IIDP centers that will result in minimal loss of quality or quantity of shipped islets (as compared to control islets remaining at the IIDP center). The objective of this Standard Operating Procedure (SOP) is to be a model for site-specific SOPs that define the method for quantitative determination of insulin released after glucose stimulation for proving the potency of the human islet preparation shipped by the IIDP.GSIR Overview:After overnight culture at 37°C (12 – 24 hrs), human islets are incubated with media containing a relatively lowconcentration of glucose (2.8 mM) and a sample of the supernatant is taken. Then the same islets are incubatedwith media containing a higher glucose concentration (28 mM) and a sample of the supernatant is taken. Theamount of insulin present in both the supernatant samples is measured using a commercially available HumanInsulin ELISA kit. The stimulation index is calculated by dividing insulin concentration of the supernatantfrom the 28 mM glucose incubation by the insulin concentration of the supernatant from the 2.8 mM glucoseincubation.Reference:Purified Human Pancreatic Islets, Glucose Stimulated Insulin Release Determination by ELISA; DAIT, NIAID, NIH SOP Appendix, Document No. 3104, A03, Revision Number 03; Effective Date: 25 Oct 2010; Supersedes Date: 08 June 2010.

Proper citation: Integrated Islet Distribution Program 2020. Potency Test: Glucose Stimulated Insulin Release Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bc3siyne Copy   

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Authors: John Tyson
Group: High molecular weight DNA extraction from all kingdoms, Long Read Club
Summary: We have been playing quite a bit with native genomic DNA sequencing for a project we have running on Epigenetic modifications of Rat models of neurological disease. While we are still rapidly iterating on tweaks etc. to some new protocols around increasing our production efficiency of ultra-long reads (100kb+) using LSK109 ligation kit components and PEG/NaCl mediated DNA precipitation, we are settling into a workflow now that I think is worth sharing and has been generating very good results for us. That said remember this is a little “wild west” and perhaps for the more adventurous at this stage, use at your own risk etc. etc. and perhaps help with some tweaks of your own ;o). I’ll say now that I have been optimizing this on a very easy Rat cell line grown in culture and realise some people don’t have this luxury, however I think this is a very good jumping off point and allowed us to iterate on a consistent starting material and will myself be venturing off into tissue extractions now.There are currently three main areas to highlight and detail changes within a sample to sequence workflow that we think has allowed us to push the efficiency and yields of sequence data out. Firstly DNA extractions using Phenol/Chloroform and spooling out of HWM DNA to generate the starting DNA sample.  Secondly input material shearing and modifications to the LSK109 ligation protocol that have yielded high efficiency libraries, and production of greater ultra-long reads numbers. Lastly the use of flowcell refueling and DNAseI clearing of the flowcell surface to remove “blocking” DNA and allow fresh library addition after a surface “reset”.I’m detailing all we have here is an attempt to allow people to fully understand what is going on as much as possible and so we can all use this information to make intelligent changes to the protocols etc. if you are so inclined. Commercial “black box kits” and solutions while great for defined on mass purpose and reproducibility make this process more opaque and often overly complex and expensive. A good example of this is the addition of just NaCl after the ligation reaction to precipitate the adapted DNA showing how knowing what you have provides simplification and economic sense for the end user in some situations, but more on that later……Final thoughts….One of my interests around nanopore sequencing and moving sequencing back to small labs and beyond with the MinION device is also mitigating the sample preparation costs while not sacrificing performance. Using cheap needles, Polyethylene Glycol, salt and old school molecular biology techniques we are seeing uncompromising performance for both yield and read lengths that I think even the “big” guys will use :o)). I will reproduce this post and methods at www.longreadclub.org soon so they will be easier to find and develop going forward, and we will try and provide protocols on www.protocols.io as well.Things on the to do list include:Further optimisation of PEG/salt parameters for size selection / short read elimination at different stages of library preparation.Refinement of shearing to provide increased 100kb+ reads from HMW DNALook at replacing Phenol/Chloroform with salting out in initial HMW Genomic DNA preparation.Who knew that PEG and salt would be a route to cheap ultra-long reads, we all just need to keep tweaking away in an open fashion and who knows where we can get. Anyway that’s enough from me for now, happy Nanopore adventuring!

Proper citation: John Tyson 2020. Rocky Mountain adventures in Genomic DNA sample preparation, ligation protocol optimisation / simplification and Ultra long read generation. protocols.io https://dx.doi.org/10.17504/protocols.io.7euhjew Copy   

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Authors: Michelle E. Favre, Valerie Lim, Michael J. Falvo, Jorge M. Serrador
Summary: Cerebrovascular reactivity and cerebral autoregulation are two major mechanisms that regulate cerebral blood flow. Both mechanisms are typically assessed in either the supine or seated postures, but the effects of body position and sex differences remain unclear. This study examined the effects of body posture (supine vs. seated vs. standing) on cerebrovascular reactivity during hyper and hypocapnia and on cerebral autoregulation during spontaneous and slow-paced breathing in healthy men and women using transcranial Doppler ultrasonography of the middle cerebral artery. Results indicated significantly improved cerebrovascular reactivity in the supine compared with seated and standing postures (supine= 3.45±0.67, seated= 2.72±0.53, standing= 2.91±0.62%/mmHg, PP. These data highlight the importance of making comparisons within the same body position to ensure there is not a confounding effect of posture.

Proper citation: Michelle E. Favre, Valerie Lim, Michael J. Falvo, Jorge M. Serrador 2020. Cerebrovascular Reactivity and Cerebral Autoregulation are Improved in the Supine Posture Compared to Upright in Healthy Men and Women. protocols.io https://dx.doi.org/10.17504/protocols.io.bb2siqee Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Artificial Cerebrospinal Fluid III (ACSF.III) is used for applications including tissue bath solution during electrophysiological recording.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Artificial Cerebrospinal Fluid III (ACSF.III). protocols.io https://dx.doi.org/10.17504/protocols.io.bdpci5iw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the steps for processing post-mortem human brain, starting from whole brain or a single hemisphere.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Proper citation: Allen Institute for Brain Science 2020. Human Post Mortem Brain Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bf4ajqse Copy   

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Authors: Angel Justiz-Vaillant, Norma McFarlane-Anderson
Group: University of the West Indies, [email protected]
Summary: This SpLG ELISA can be used to detect specific antibodies in various animal species including human, mouse, rat, dog, rabbit, chicken, monkey, pig and hamster [1].1. Kihlberg BM, Sjöbring U, Kastern W, Björck L. Protein LG: a hybrid molecule with unique immunoglobulin binding properties.J Biol Chem. 1992;267(35):25583-25588.

Proper citation: Angel Justiz-Vaillant, Norma McFarlane-Anderson 2020. Enzyme linked immunosorbent assay for investigating the binding of protein-LG (SpLG) to immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjpmkmk6 Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the standard protocol for routine insulin tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or for 6 hours.

Proper citation: Henri Brunengraber 2019. Case - Intraperitoneal Insulin Tolerance Test. protocols.io https://dx.doi.org/10.17504/protocols.io.yefftbn Copy   

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Authors: Tjusls China

Proper citation: Tjusls China 2019. 18 Monitoring in living bacterial cells by UV-Vis spectroscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.7y8hpzw Copy   

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