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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Proper citation: Matthew Sullivan 2016. Titration of AmPure XP Beads for Removal of Fragments . protocols.io dx.doi.org/10.17504/protocols.io.c52y8d Copy
Authors: Vivian Liu
Summary: This brief protocol shows you how easy it is to get shRNA from demostrated siRNA sequence. With these self-designed shRNA, I've got more than 10 genes knockdown from mammalian cell lines, including mouse embryonic stem cells. Believe it or not, using siRNA sequence from Dharmacon will guarantee you the success.
Proper citation: Vivian Liu 2015. From siRNA to shRNA. protocols.io dx.doi.org/10.17504/protocols.io.dsr6d5 Copy
Authors: John Davis Coakley
Summary: How to prepare PBMCs for Stimulation experiment
Proper citation: John Davis Coakley 2019. Peripheral Blood Mononuclear Cells Preparation. protocols.io dx.doi.org/10.17504/protocols.io.6zyhf7w Copy
Authors: N.J. Hillson
Proper citation: N.J. Hillson 2019. One Part CPEC and quick change. protocols.io dx.doi.org/10.17504/protocols.io.6vdhe26 Copy
Authors: Irena Kaczmarska, James M. Ehrman
Summary: This protocol describes the tracing of newly deposited silica using PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole) in reproductive studies of diatoms.
Proper citation: Irena Kaczmarska, James M. Ehrman 2017. PDMPO protocol and optional fixation steps for tracing newly deposited silica in plagiogrammacean auxospores and their progeny. protocols.io dx.doi.org/10.17504/protocols.io.huub6ww Copy
Authors: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong
Group: GigaScience Press, BGI
Summary: Illumina GAII Library Construction and Sequencing for RNA Seq
Proper citation: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong 2019. Illumina GAII Library Construction and Sequencing for RNA Seq. protocols.io dx.doi.org/10.17504/protocols.io.38mgru6 Copy
Authors: Anika Wiegard, Ilka Maria Axmann
Group: Axmann Lab
Summary: SDS-PAGE using gels with low crosslinking of acrylamide and bisacrylamide. This protocol can be used to separate phosphorylation forms of KaiC proteins.
Proper citation: Anika Wiegard, Ilka Maria Axmann 2017. LowC SDS-PAGE. protocols.io dx.doi.org/10.17504/protocols.io.gysbxwe Copy
Authors: Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Rachele Cesaroni 2019. Quick staining procedure of nuclei in Euplotes using DAPI. protocols.io dx.doi.org/10.17504/protocols.io.2ajgacn Copy
Authors: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the, the thraustochytrids. Limnol. Oceanogr.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct
detection and enumeration of the fungilike marine protists, the thraustochytrids. Limnol.
Oceanogr. 38(1): 182-187.
Proper citation: This protocol is modified from: Raghukumar S, Schaumann K. 1993. An epifluorescence microscopy method for direct detection and enumeration of the, the thraustochytrids. Limnol. Oceanogr. 2017. Acriflavine Direct Detection Technique for Labyrinthulomycetes. protocols.io dx.doi.org/10.17504/protocols.io.hfib3ke Copy
Authors: Scott Daniel
Group: Hurwitz Lab
Summary: Overview:1. Filter DNA reads by quality and host (e.g. for mouse gut bacteria we would filter low-quality reads AND reads that belong to mice)2. Align DNA reads (fastq, fasta) to Patric database (bacteria genomes) with taxoner (a bowtie2-based linux program) >> Get bacterial genomes3. Filter out low-quality alignments >> Bacterial genomes4. Filter RNA reads by quality5. Align RNA reads to bacterial genomes from step 3 >> RNA counts of genes6. Get pathway information >> RNA counts per KEGG pathwayIf you have treatment groups, we will be able to see changes in bacterial abundance as well as changes to RNA counts
Proper citation: Scott Daniel 2018. Combined Metagenomic / Metatranscriptomic Pipeline for Host-associated Microbiomes. protocols.io dx.doi.org/10.17504/protocols.io.d7m9k5 Copy
Authors: Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time RT-PCR using an MGB probe, this assay detects Japanese encephalitis virus (JEV) from human and mosquito samples.The assay targets the 3'UTR region of known JEV strains.
Proper citation: Judy Northill, Mitchell Finger, Michael Lyon, Ian Mackay 2018. Japanese encephalitis virus real-time RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.r23d8gn Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. It is intended for use in gravity or pressure flow columns, and batch purifications. NEBExpress Ni Resin is comprised of a highly uniform and stable chemical-tolerant resin, pre-charged with nickel ions on the matrix surface. It is resistant to a wide range of chemicals, including NaOH, EDTA, DTT and β-Mercaptoethanol.Purification of ≥10 mg His-tagged protein per 1 ml of resinIntended for use in gravity or pressure flow columns, and batch purificationsHigh specific binding of His-tagged proteins yielding purities of >95%Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagentsIsolation and purification of His-tagged fusion proteins under native or denaturing conditions
Proper citation: New England Biolabs 2020. NEBExpress Ni Resin Batch Binding Typical Protocol (NEB #S1428). protocols.io dx.doi.org/10.17504/protocols.io.bfd5ji86 Copy
Authors: Julie Huber, Caroline Fortunato
Group: Center for Dark Energy Biosphere Investigations, Huber Lab
Summary: This is the protocol to extract RNA from Sterivex filters from RNA-SIP experiments carried out with seawater, vent fluids, etc.
Proper citation: Julie Huber, Caroline Fortunato 2017. RNA Extraction Protocol from RNA-SIP Experiments. protocols.io dx.doi.org/10.17504/protocols.io.iuycexw Copy
Authors: Mark Moosburner, Andrew Allen
Group: A.E. Allen Lab
Summary: To ensure Cas9 activity upon episomal transformation, the 3’ end of the Cas9 coding sequencing was transcriptionally fused to an antibiotic selectable marker, shble, by the 2A peptide. The Cas9-2A-shble construct was cloned into a Phaeodactylum episome that included a bacterial expression cassette for a red fluorescent protein (RFP). The RFP was also flanked by two BsaI restriction digest sites. The RFP vector with Cas9 was used as a cloning vector to assembly one or more sgRNA Phaeodactylum expression cassettes and a single LacZ bacterial expression cassette in place of the RFP. Upon successful assembly, the correct colonies appear blue on selective agar plates while incorrect colonies appear either red or colorless. The golden gate assembly protocol used to synthesize the episome (g51092-2, g24739-A) is as follows:
Proper citation: Mark Moosburner, Andrew Allen 2019. GG2 - CRISPR-Cas9 episome cloning using red-blue screening for Phaeodactylum tricornutum. protocols.io dx.doi.org/10.17504/protocols.io.4acgsaw Copy
Authors: Xavier Contreras, Rosemary Kiernan
Summary: Adapted from Dignam JD, Lebovitz RM, Roeder RG. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11(5):1475-89. PMID:6828386
Proper citation: Xavier Contreras, Rosemary Kiernan 2018. High Salt Nuclear Extract Preparation. protocols.io dx.doi.org/10.17504/protocols.io.kh2ct8e Copy
Authors: Yang Li
Summary: Experimental ProceduresCell culture, reagents, and transfectionCells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, Novato, CA), 100 U/ml penicillin and 100 mg/ml streptomycin. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. MG132 and doxycycline were obtained commercially. Fibroblasts from control individuals and HD patients with the Htt47Q and Htt68Q expansions were obtained from Dr. Boxun Lu’s lab in Fudan University.In brief, primary cortex neurons were isolated from appropriately timed pregnant female (16.5 days). Using forceps, we isolated cortex from each hemisphere of mouse brains under microscope. Once the cortex regions were isolated, they were cut into approximately 10 smaller pieces for digestion (1 ml prewarmed Trypsin, at 37℃ for 20 min). Then serum was added to inactivate trypsin. After aspirating the supernatant, we added 1 ml prewarmed DNase solution for 10 min at 37℃. Then, the cells were dissociated by pipetting up and down, and all supernatant were transferred to new tubes for subsequent centrifugation (800xg for 5 min). We resuspended cell pellet gently and plated them on coated dishes (Poly-D-Lysine, BD, #354210). The neuronal culture medium was changed every 2 days. After about 14 days of neuronal culture, we performed transient transfection of Htt97Q-EGFP (1 mg) or/and siWDR81 (100 pmol, three times at an interval of 24 h) using Lipofectamine MessengerMAX (Thermo Fisher Scientific) as manufacturing introduction. Immunostaining and confocal microscopyCells grown on coverslips were fixed in 4% paraformaldehyde followed by permeabilization with 0.05% saponin. After extensive washing with phosphate buffered saline (PBS), cells were incubated with primary antibodies in PBS containing 5% BSA at 4ºC overnight. Cells were washed extensively again and incubated with secondary antibodies for 1 h at room temperature. Following another round of thorough washing, cells were sealed on slides for microscopy analysis. For live cell imaging, cells were grown in confocal dishes (Glass Bottom Dish, In Vitro Scientific, Sunnyvale, California, USA). Samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer.Generation of cell lines stably expressing Tet-on Htt97Q-GFPHeLa cells stably expressing pcDNA6/TR (kindly provided by Dr. Quan Chen) were transfected with pcDNA4-Htt97Q-GFP. 24 h later, cells were subjected to selection for 2-3 weeks in medium supplemented with blasticidin (5 mg/ml) and zeocin (100 mg/ml). Single colonies were picked and propagated further. The expression of Htt97Q-GFP was determined by adding doxycycline (1 mg/ml) and observed by fluorescence microscopy.Htt polyQ clearance assayTo assess the effect of siRNA knockdown of WDR81 on clearance of Htt polyQ inclusions, HeLa cells stably expressing Tet-on Htt97Q-EGFP were transfected twice at an interval of 24 h with siRNA oligos against WDR81. 12 h afterthe second siRNA transfection, Htt97Q-EGFP expression was induced with doxycycline (1 mg/ml). The medium was changed 12 h later to remove the doxycycline and the cells were split into several dishes for the remaining siRNA treatments. Time-course quantification of polyQ foci was performed 12 h after the induction of Htt97Q-EGFP expression. To rescue WDR81 siRNA-induced accumulation of polyQ foci, cells were first treated with control or WDR81 siRNA, then 3 mg of vector expressing siRNA-resistant WDR81 were transfected into the same cells simultaneous with the induction of Htt97Q-EGFP expression by doxycycline. siRNA treatments were performed twice more as above and polyQ foci were scored 48 h post induction of Htt97Q-EGFP expression.C. elegans geneticsC. elegans Bristol strain N2 was used as wild type. sorf-2(tm5210) deletion mutants were provided by Dr. Shohei Mitani (Tokyo Women’s Medical University, Japan). The integrated arrays bpIs267 and baIn1 were kindly provided by Dr. Hong Zhang (Institute of Biophysics, CAS, China) and Dr. Guy Caldwell (University of Alabama, Tuscaloosa). C. elegans cultures, genetic crosses, and generation of transgenic animals were performed according to standard procedures (Brenner 1974).Human subjects and IHC procedureBrain samples from 12 control individuals and 12 patients with indicated neurodegenerative diseases (AD, n=4; HD, n=3; PD, n=5) were obtained from theChinese Brain Bank of Zhejiang University (Table 1). The samples are paraffin-embedded sections of hippocampus and cortex of frontal lobe. The controls and patients were strictly paired according to parameters including age, gender, reason of death and phase of disease. All procedures were performed according to protocols approved by Human Ethics Committees at both Fudan University and Zhejiang University.Paraffin-embedded sections were dewaxed, rehydrated, and rinsed in PBS. After being boiled for 15 min in 0.01 mol/liter sodium citrate buffer (pH 6.0), sections were blocked in 5% guinea pig (Gp) or rabbit pre-immune serum in PBS for 1 h at room temperature and then incubated overnight with WDR81 antibody (Gp, 1:200) or p62 antibody (rabbit, 1:1000). Sections were then incubated in horseradish peroxidase-conjugated secondary antibody (1:200) at room temperature for 3 hours. After 4 washes in PBS, 3,3-diaminobenzidine (DAB) was added for 1 min at room temperature. Sections were counterstained with hematoxylin for 2 min. The IHC pictures were obtained by Nikon microscope (Ts2R). Results were calculated by software Image J and NIS-Element, BR. 3.00 (Nikon). For quantification measurements, images were randomly acquired from the hippocampus and cortex of frontal lobe and analyzed by researchers in a double-blind manner.Expression vectorsThe mammalian, bacterial and C. elegans expression vectors listed below were constructed using standard protocols. Expression vectorsGene(cDNA)Insertion siteBackbonepmCherry-c1-WDR81WDR81XhoI/BamHIpmCherry-c1pEGFP-c1-WDR81WDR81XhoI/XbaIpEGFP-c1pEGFP-c1-WDR81(1-650)WDR81 XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(730-1440)WDR81(730-1440)XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(1637-1940)WDR81(1637-1940)XhoI/BamHIpEGFP-c1pcDNA4-Htt97Q-GFPHtt97Q-GFPEcoRI/XhoIpcDNA4-TO-myc-HisP Y37A1B.5Myc-sorf-2sorf-2 ORF with MycXmaIpPD49.26P Y37A1B.5WDR81WDR81KpnI/EcoRVpPD49.26Pdat-1 sorf-2dat-1 promoter and sorf-2 ORFPstI/XmaI for Pdat-1 XmaI /NcoI for sorf-2pPD95.77Pdat-1 WDR81dat-1 promoterSphI/BamHIP Y37A1B.5WDR81pmCherry-c1-ATG5Atg5XhoI/BamHIpmCherry-c1pmCherry-c1-ATG12Atg12XhoI/BamHIpmCherry-c1The following vector were kindly provided by other scientists: pcDNA3.1-Htt97Q-EGFP (Dr. Xiaojiang Li, Institute of Genetics and Developmental Biology, CAS, China). Small interfering RNAs (siRNAs) The oligos used for siRNA knockdown of WDR81 were as described previously [14]. Other oligos were as follows: p62: 5’-GCATTGAAGTTGATATCGAT-3’[37];ATG12: 5’-AUGAGCUUCAAUUGCAUCCtt-3’ [38] ; Control siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’.Cells were transfected with 100 pmol siRNA oligos twice at an interval of 24 h. Cells were subjected to further analysis 24 h after the last transfection. For the transfection of primary neurons, we performed the assay using Lipofectamine MessengerMax reagent (Invitrogen).Quantitative reverse transcription–polymerase chain reaction (qPCR)Total RNA was extracted using Trizol (Invitrogen) and chloroform. 2 μg of RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (Promega, Madison, Wisconsin, USA). qPCR reactions were carried out on an MX3000P system (Agilent Technologies, Santa Clara, CA).AntibodiesWDR81 antibodies were generated in guinea pigs and rabbits by injecting purified recombinant GST-WDR81(332-604) and purchased from ABclonal (#A12780). GFP and mCherry antibodies were generated in guinea pigs or rabbits by injecting recombinant proteins. Rabbit polyclonal antibodies to p62 were purchased from Medical & Biological Laboratories (MBL, Nagoya, Japan). Mouse monoclonal antibodies against ATG12 were purchased from MBL. Mouse monoclonal antibodies to b-actin and GFP were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3-, and FITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).Western blotting and immunoprecipitationTo analyze levels of proteins of interest, cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1% TritonX-100, 1 mM phenylmethanesulfonyl fluoride (PMSF)) or RIPA buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1 mM PMSF) containing one Complete Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland). Cell lysates were spun down at 12000 rpm for 10 min. 50 g of supernatants were resolved onsodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted with the indicated antibodies. b-actin was used as the loading control.To determine the interaction of endogenous WDR81 with autophagy factors or Htt polyQ-associated proteins, HeLa cells with or without Htt97Q-EGFP expression were lysed in ice-cold Triton X-100 buffer containing one Complete Protease Inhibitor Cocktail Tablet (Roche). In the case of Htt97Q-EGFP IP, the cell lysates were cleared by centrifugation at 500xg as described by Filimonenko et al. [12]. Cleared cell lysates were first mixed with antibodies to WDR81 or GFP (~5 mg) for 4 h followed by incubation with protein A agarose beads (10 ml) (GE Healthcare) overnight at 4°C. Analysis of precipitated proteins was performed as above.MTT assay for cell viabilityThe MTT kit was purchased from Promega (#G4002). Cells were cultured in 96-well plates with DMEM containing 10% FBS. After various treatments, 15 mL of dye solution was added into each well. Then the plates were incubated at 37℃ for 1.5 hours in a humidified CO2 incubator. Stop solution (100 mL) was added into each well, and the absorbance was recorded at 570 nm using plate reader. 630 nm was used as a reference wavelength.Transmission electron microscopyCells were fixed for 2.5 h at 4°C with 0.1 M PBS containing 2.5% glutaraldehyde. Then, the fixed samples were rinsed with PBS and 1% OsO4 for 0.5 h at 4°C. The samples were rinsed with distilled water and dehydrated by sequential incubation with an acetone series (30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%, 5 min each). After that, samples were infiltrated with Araldite 502/Embed 812 by gradually increasing the concentration of acetone (25% and 50%, 20 min; 75%, 30 min; 100%, 20 h) and then polymerized at 60°C for 70 h. Embedded samples were sectioned using an UC6 ultramicrotome (Leica Biosystems) equipped with a 45° diamond knife (Diatome) to obtain 70-nm ultrathin sections. The grids were stained at room temperature with 2% aqueous uranyl acetate (10 min) and Reynolds lead citrate (5 min) before imaging. Imaging was performed at 80 kV on a JEM-1400 (JEOL) transmission electron microscope.Statistics and reproducibilityData were analyzed with Prism (GraphPad software). Statistical analyses were performed using t-tests or ANOVA. *P0.05 was considered not significant (NS). Experimental ProceduresCell culture, reagents, and transfectionCells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, Novato, CA), 100 U/ml penicillin and 100 mg/ml streptomycin. Transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. MG132 and doxycycline were obtained commercially. Fibroblasts from control individuals and HD patients with the Htt47Q and Htt68Q expansions were obtained from Dr. Boxun Lu’s lab in Fudan University.In brief, primary cortex neurons were isolated from appropriately timed pregnant female (16.5 days). Using forceps, we isolated cortex from each hemisphere of mouse brains under microscope. Once the cortex regions were isolated, they were cut into approximately 10 smaller pieces for digestion (1 ml prewarmed Trypsin, at 37℃ for 20 min). Then serum was added to inactivate trypsin. After aspirating the supernatant, we added 1 ml prewarmed DNase solution for 10 min at 37℃. Then, the cells were dissociated by pipetting up and down, and all supernatant were transferred to new tubes for subsequent centrifugation (800xg for 5 min). We resuspended cell pellet gently and plated them on coated dishes (Poly-D-Lysine, BD, #354210). The neuronal culture medium was changed every 2 days. After about 14 days of neuronal culture, we performed transient transfection of Htt97Q-EGFP (1 mg) or/and siWDR81 (100 pmol, three times at an interval of 24 h) using Lipofectamine MessengerMAX (Thermo Fisher Scientific) as manufacturing introduction. Immunostaining and confocal microscopyCells grown on coverslips were fixed in 4% paraformaldehyde followed by permeabilization with 0.05% saponin. After extensive washing with phosphate buffered saline (PBS), cells were incubated with primary antibodies in PBS containing 5% BSA at 4ºC overnight. Cells were washed extensively again and incubated with secondary antibodies for 1 h at room temperature. Following another round of thorough washing, cells were sealed on slides for microscopy analysis. For live cell imaging, cells were grown in confocal dishes (Glass Bottom Dish, In Vitro Scientific, Sunnyvale, California, USA). Samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer.Generation of cell lines stably expressing Tet-on Htt97Q-GFPHeLa cells stably expressing pcDNA6/TR (kindly provided by Dr. Quan Chen) were transfected with pcDNA4-Htt97Q-GFP. 24 h later, cells were subjected to selection for 2-3 weeks in medium supplemented with blasticidin (5 mg/ml) and zeocin (100 mg/ml). Single colonies were picked and propagated further. The expression of Htt97Q-GFP was determined by adding doxycycline (1 mg/ml) and observed by fluorescence microscopy.Htt polyQ clearance assayTo assess the effect of siRNA knockdown of WDR81 on clearance of Htt polyQ inclusions, HeLa cells stably expressing Tet-on Htt97Q-EGFP were transfected twice at an interval of 24 h with siRNA oligos against WDR81. 12 h afterthe second siRNA transfection, Htt97Q-EGFP expression was induced with doxycycline (1 mg/ml). The medium was changed 12 h later to remove the doxycycline and the cells were split into several dishes for the remaining siRNA treatments. Time-course quantification of polyQ foci was performed 12 h after the induction of Htt97Q-EGFP expression. To rescue WDR81 siRNA-induced accumulation of polyQ foci, cells were first treated with control or WDR81 siRNA, then 3 mg of vector expressing siRNA-resistant WDR81 were transfected into the same cells simultaneous with the induction of Htt97Q-EGFP expression by doxycycline. siRNA treatments were performed twice more as above and polyQ foci were scored 48 h post induction of Htt97Q-EGFP expression.C. elegans geneticsC. elegans Bristol strain N2 was used as wild type. sorf-2(tm5210) deletion mutants were provided by Dr. Shohei Mitani (Tokyo Women’s Medical University, Japan). The integrated arrays bpIs267 and baIn1 were kindly provided by Dr. Hong Zhang (Institute of Biophysics, CAS, China) and Dr. Guy Caldwell (University of Alabama, Tuscaloosa). C. elegans cultures, genetic crosses, and generation of transgenic animals were performed according to standard procedures (Brenner 1974).Human subjects and IHC procedureBrain samples from 12 control individuals and 12 patients with indicated neurodegenerative diseases (AD, n=4; HD, n=3; PD, n=5) were obtained from theChinese Brain Bank of Zhejiang University (Table 1). The samples are paraffin-embedded sections of hippocampus and cortex of frontal lobe. The controls and patients were strictly paired according to parameters including age, gender, reason of death and phase of disease. All procedures were performed according to protocols approved by Human Ethics Committees at both Fudan University and Zhejiang University.Paraffin-embedded sections were dewaxed, rehydrated, and rinsed in PBS. After being boiled for 15 min in 0.01 mol/liter sodium citrate buffer (pH 6.0), sections were blocked in 5% guinea pig (Gp) or rabbit pre-immune serum in PBS for 1 h at room temperature and then incubated overnight with WDR81 antibody (Gp, 1:200) or p62 antibody (rabbit, 1:1000). Sections were then incubated in horseradish peroxidase-conjugated secondary antibody (1:200) at room temperature for 3 hours. After 4 washes in PBS, 3,3-diaminobenzidine (DAB) was added for 1 min at room temperature. Sections were counterstained with hematoxylin for 2 min. The IHC pictures were obtained by Nikon microscope (Ts2R). Results were calculated by software Image J and NIS-Element, BR. 3.00 (Nikon). For quantification measurements, images were randomly acquired from the hippocampus and cortex of frontal lobe and analyzed by researchers in a double-blind manner.Expression vectorsThe mammalian, bacterial and C. elegans expression vectors listed below were constructed using standard protocols. Expression vectorsGene(cDNA)Insertion siteBackbonepmCherry-c1-WDR81WDR81XhoI/BamHIpmCherry-c1pEGFP-c1-WDR81WDR81XhoI/XbaIpEGFP-c1pEGFP-c1-WDR81(1-650)WDR81 XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(730-1440)WDR81(730-1440)XhoI/BamHIpEGFP-c1pEGFP-c1-WDR81(1637-1940)WDR81(1637-1940)XhoI/BamHIpEGFP-c1pcDNA4-Htt97Q-GFPHtt97Q-GFPEcoRI/XhoIpcDNA4-TO-myc-HisP Y37A1B.5Myc-sorf-2sorf-2 ORF with MycXmaIpPD49.26P Y37A1B.5WDR81WDR81KpnI/EcoRVpPD49.26Pdat-1 sorf-2dat-1 promoter and sorf-2 ORFPstI/XmaI for Pdat-1 XmaI /NcoI for sorf-2pPD95.77Pdat-1 WDR81dat-1 promoterSphI/BamHIP Y37A1B.5WDR81pmCherry-c1-ATG5Atg5XhoI/BamHIpmCherry-c1pmCherry-c1-ATG12Atg12XhoI/BamHIpmCherry-c1The following vector were kindly provided by other scientists: pcDNA3.1-Htt97Q-EGFP (Dr. Xiaojiang Li, Institute of Genetics and Developmental Biology, CAS, China). Small interfering RNAs (siRNAs) The oligos used for siRNA knockdown of WDR81 were as described previously [14]. Other oligos were as follows: p62: 5’-GCATTGAAGTTGATATCGAT-3’[37];ATG12: 5’-AUGAGCUUCAAUUGCAUCCtt-3’ [38] ; Control siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’.Cells were transfected with 100 pmol siRNA oligos twice at an interval of 24 h. Cells were subjected to further analysis 24 h after the last transfection. For the transfection of primary neurons, we performed the assay using Lipofectamine MessengerMax reagent (Invitrogen).Quantitative reverse transcription–polymerase chain reaction (qPCR)Total RNA was extracted using Trizol (Invitrogen) and chloroform. 2 μg of RNA was used as template to generate cDNAs using the ImProm-II Reverse Transcription system (Promega, Madison, Wisconsin, USA). qPCR reactions were carried out on an MX3000P system (Agilent Technologies, Santa Clara, CA).AntibodiesWDR81 antibodies were generated in guinea pigs and rabbits by injecting purified recombinant GST-WDR81(332-604) and purchased from ABclonal (#A12780). GFP and mCherry antibodies were generated in guinea pigs or rabbits by injecting recombinant proteins. Rabbit polyclonal antibodies to p62 were purchased from Medical & Biological Laboratories (MBL, Nagoya, Japan). Mouse monoclonal antibodies against ATG12 were purchased from MBL. Mouse monoclonal antibodies to b-actin and GFP were purchased from Sigma-Aldrich (St. Louis, MO). HRP-, Cy3-, and FITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).Western blotting and immunoprecipitationTo analyze levels of proteins of interest, cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1% TritonX-100, 1 mM phenylmethanesulfonyl fluoride (PMSF)) or RIPA buffer (20 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1 mM PMSF) containing one Complete Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland). Cell lysates were spun down at 12000 rpm for 10 min. 50 g of supernatants were resolved onsodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted with the indicated antibodies. b-actin was used as the loading control.To determine the interaction of endogenous WDR81 with autophagy factors or Htt polyQ-associated proteins, HeLa cells with or without Htt97Q-EGFP expression were lysed in ice-cold Triton X-100 buffer containing one Complete Protease Inhibitor Cocktail Tablet (Roche). In the case of Htt97Q-EGFP IP, the cell lysates were cleared by centrifugation at 500xg as described by Filimonenko et al. [12]. Cleared cell lysates were first mixed with antibodies to WDR81 or GFP (~5 mg) for 4 h followed by incubation with protein A agarose beads (10 ml) (GE Healthcare) overnight at 4°C. Analysis of precipitated proteins was performed as above.MTT assay for cell viabilityThe MTT kit was purchased from Promega (#G4002). Cells were cultured in 96-well plates with DMEM containing 10% FBS. After various treatments, 15 mL of dye solution was added into each well. Then the plates were incubated at 37℃ for 1.5 hours in a humidified CO2 incubator. Stop solution (100 mL) was added into each well, and the absorbance was recorded at 570 nm using plate reader. 630 nm was used as a reference wavelength.Transmission electron microscopyCells were fixed for 2.5 h at 4°C with 0.1 M PBS containing 2.5% glutaraldehyde. Then, the fixed samples were rinsed with PBS and 1% OsO4 for 0.5 h at 4°C. The samples were rinsed with distilled water and dehydrated by sequential incubation with an acetone series (30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%, 5 min each). After that, samples were infiltrated with Araldite 502/Embed 812 by gradually increasing the concentration of acetone (25% and 50%, 20 min; 75%, 30 min; 100%, 20 h) and then polymerized at 60°C for 70 h. Embedded samples were sectioned using an UC6 ultramicrotome (Leica Biosystems) equipped with a 45° diamond knife (Diatome) to obtain 70-nm ultrathin sections. The grids were stained at room temperature with 2% aqueous uranyl acetate (10 min) and Reynolds lead citrate (5 min) before imaging. Imaging was performed at 80 kV on a JEM-1400 (JEOL) transmission electron microscope.Statistics and reproducibilityData were analyzed with Prism (GraphPad software). Statistical analyses were performed using t-tests or ANOVA. *P0.05 was considered not significant (NS).
Proper citation: Yang Li 2021. protocols in WDR81 paper. protocols.io dx.doi.org/10.17504/protocols.io.brg5m3y6 Copy
Authors: Kelsey Miller
Summary: Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher than 50% then please follow protocol B.The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Proper citation: Kelsey Miller 2016. MojoSort™ Human CD45 Nanobeads Protocol B. protocols.io dx.doi.org/10.17504/protocols.io.e22bgge Copy
Authors: Maximilian Dietsch, Vera Wewer
Group: Axmann Lab, CyanoWorld
Summary: In this protocol, the extraction of naturally occurring as well as heterologously synthesized triterpenes in Synechocystis and R. capsulatus are described
Proper citation: Maximilian Dietsch, Vera Wewer 2018. Triterpene extraction protocol from Synechocystis sp. PCC6803 and R. capsulatus. protocols.io dx.doi.org/10.17504/protocols.io.j6tcren Copy
Authors: Sid Roy
Group: GenapSys
Summary: User guide to prepare your libraries for sequencing on the GenapSys Sequencer.
Proper citation: Sid Roy 2020. Sequencing Protocol (GenapSys). protocols.io dx.doi.org/10.17504/protocols.io.bkxfkxjn Copy
Authors: Bryce Killingsworth, Joshua Welsh, Tim Traynor, Jennifer Jones
Group: Translational Nanobiology Section
Summary: This is a generic protocol for using the Spectradyne nCS1 resestive pulse sensing instrument to measure the diameter distribution and concentration of nanoparticles.
Proper citation: Bryce Killingsworth, Joshua Welsh, Tim Traynor, Jennifer Jones 2020. Spectradyne nCS1: Sample measurement and device maintenence protocol. protocols.io dx.doi.org/10.17504/protocols.io.bf9pjr5n Copy
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