Protocols   Select Another Resource Report Type

Authors: Elisangela Bressan, Ashutosh Dhingra, Stella Donato, Peter Heutink
Summary: The derivation of human induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons presents an exciting opportunity to access a large number of patients-specific cells in vitro, to model disease, perform target screenings, and test drug candidates. Previously published small molecule-based protocols are straightforward, implementable in automated cell culture systems (Dhingra et al. J Vis Exp. 162, 2020), and suitable for the differentiation of large sets of cell lines (Bressan et al. Protocols.io, 2020) at relatively low cost and working time. However, the derivation of mDA from a large set of human iPSC lines shows variations in differentiation efficiency between lines. In addition, the current protocols produce heterogeneous cell populations in which only a small subset represents the cells of interest. To address these issues, we optimized a previously established mDA neuron differentiation protocol (Kriks et al., Nature 480, 547–551, 2011) by: (1) adjusting the SMAD inhibition to improve and achieve more homogeneous neuron conversion between iPSC lines; (2) reinforcing the WTN signaling activation to trigger more efficient midbrain floor plate induction and conversion into mDA neurons, and (3) applying the DNA cross-linker Mitomycin-C to eliminate remaining proliferating cells. The optimized mDA neuron derivation protocol presented here resulted in more homogeneous differentiation between iPSC lines, higher yields of neurons with higher proportion of mDA neurons, and completely elimination of contaminating proliferative cells. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Elisangela Bressan, Ashutosh Dhingra, Stella Donato, Peter Heutink 2021. Optimized Derivation of Midbrain Dopaminergic Neurons from iPSCs for research application. protocols.io https://dx.doi.org/10.17504/protocols.io.bsq5ndy6 Copy   

•   Source:

Authors: Predrag Noveski, Marija Terzic, Marija Vujovic, Maja Kuzmanovska, Emilija Sukarova Stefanovska, Dijana Plaseska-Karanfilska
Summary: Quantitative fluorescent polymerase chain reaction (QF-PCR) is reliable and efficient method for the rapid prenatal diagnosis of common fetal aneuploidies. In this protocol we first describe a procedure for isolation of DNA from amniocytes and chorionic villi with the use of commercial kit intended for the isolation of nucleic acids from viruses, which, taking into account the phenomenon of increased fragmentation of the DNA material of fetal origin, proved to be appropriate for prenatal diagnosis. (Section 1). As part of the isolation procedure, we also include a procedure for treating amniotic fluid with visible contamination (blood with possible origin from the mother).Тhe presented procedure consists of simple physical separation of the maternal blood cells from the fetal material in order to reduce the failure rate due to maternal cell contamination. .justify:after { content: ""; display:inline-block; width: 100%; } In the second part of the protocol (Section 2) we are presenting procedure for performing the QF-PCR analysis. Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies, which targeted in total 26 genomic positions. Of them, three markers (D13S258, D13S305 and D13S1817) were for analysis of the aneuploidies for the chromosome 13, four (D18S386, D18S390, D18S391 and D18S535) for the chromosome 18, four (D21S1411, D21S1414, D21S1435 and D21S1446) for the chromosome 21 and six for the analysis of the sex chromosome aneuploidies. Two of the sex chromosome markers (DXS6803 and XHPRT) amplified sequences only on chromosome X (for counting chromosome X), two (AMELX/Y and DXYS218) co-amplified sequences of both X and Y chromosomes (for counting chromosomes X and Y in male samples and chromosome X in female samples), one (TAF9B) was for co-amplification of sequences on chromosomes X and 3 (for counting chromosome X) and one (SRY) located on chromosome Y was for male sex determination. Three remaining markers (MYPT2/Y, DYS448 and CDY1/2) were used simultaneously for detection of the Y chromosome aneuploidies and microaberrations in the azoospermia factor region “c” (AZFc), located on the Y chromosome. .justify:after { content: ""; display:inline-block; width: 100%; } For the cases with inconclusive results, we used additional QF-PCR reactions specific for given chromosome (Section 3). For chromosomes 13, 18 and 21 the primers were combined in one multiplex reaction each: four STR markers for chromosome 13 (D13S742, D13S628, D13S634 and D13S631), three STR markers for chromosome 18 (D18S51, D18S1367 and D18S978) and four STR markers for chromosome 21 (D21S1437, D21S11, D21S1412 and D21S1441). For the sex chromosomes we have used three markers as separate PCR reactions (for DXS6809, DXS996 and X22) and additionaly if needed, previously published multiplex reaction [File 1] for detection of sex chromosome aneuploidies in infertile patients. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Predrag Noveski, Marija Terzic, Marija Vujovic, Maja Kuzmanovska, Emilija Sukarova Stefanovska, Dijana Plaseska-Karanfilska 2019. Quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal diagnosis of common fetal aneuploidies. protocols.io https://dx.doi.org/10.17504/protocols.io.2v9ge96 Copy   

•   Source:

Authors: Jason Stajich
Group: stajichlab

Proper citation: Jason Stajich 2014. CTAB-Urea buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.chet3d Copy   

•   Source:

Authors: Chris Cheshire

Proper citation: Chris Cheshire 2021. DNA Barcoded Hydrogel Bead Synthesis. protocols.io https:// Copy   

•   Source:

Authors: Jacob Matson, Jean Cook
Summary: Chromatin flow cytometry for MCM loading cell cycle analysis, adaptable to any antibody measurements by chromatin flow cytometry.

Proper citation: Jacob Matson, Jean Cook 2020. MCM Chromatin flow cytometry for cell cycle. protocols.io https://dx.doi.org/10.17504/protocols.io.bba8iihw Copy   

•   Source:

Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2020. MojoSort™ Human CD14+ Monocyte Isolation Kit Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbwaipae Copy   

•   Source:

Authors: Hannigan GD, Grice EA, et al.
Group: VERVE Net, Club Grice
Summary: This protocol provides methods for quality control of metagenomic data. Included is adapter trimming, quality trimming, decontamination, negative control removal, and pre-processing results. Based on the methods found in the following publication:Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: Hannigan GD, Grice EA, et al. 2016. Script P1: Pre-Processing Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.edrba56 Copy   

•   Source:

Authors: LeinU01 BRAIN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis.

Proper citation: LeinU01 BRAIN Grant 2019. Isolation of Nuelci from Adult_Human_Brain_Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.y6pfzdn Copy   

•   Source:

Authors: Adélaïde Roguet, Shuchen Feng
Group: Coronavirus Method Development Community, mclellan lab

Proper citation: Adélaïde Roguet, Shuchen Feng 2021. Nucleic acid extraction - RNeasy PowerMicrobiome Kit (Qiagen). protocols.io https://dx.doi.org/10.17504/protocols.io.bpg5mjy6 Copy   

•   Source:

Authors: J Jobu Babin
Summary: This paper reports the results of an experiment involving text-messaging and emojis in laboratory trust games executed on mobile devices. Decomposing chat logs, I find that trust increases dramatically with the introduction of emojis to one-shot games, while reciprocation increases only modestly. Skin tones embeddedin emojis impact sharing and resulting gains – to the benefit of some anddetriment to others. Both light and dark skin players trust less on receipt of a dark skin tone emoji – suggestive of statistical discrimination. In this way, computer-mediated communication leads to reduced gains for dark-skinned persons. These results highlight the complex social judgment that motivates trust in an anonymous counterpart.

Proper citation: J Jobu Babin 2020. Linguistic signaling, emojis, and skin tone in trust games. protocols.io https://dx.doi.org/10.17504/protocols.io.bf54jq8w Copy   

•   Source:

Authors: Claudia Troncone Clemente
Group: AEGIS - Madrid iGEM 2019
Summary: Our aim with this protocol is to amplify DNA. This protocol has been optimized has a general amplificationAs the quantity of DNA is exponentially increased during the performance of the selection, further modification in the numbers of cycle will be needed to be implemented.

Proper citation: Claudia Troncone Clemente 2019. PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.8nyhvfw Copy   

•   Source:

Authors: André Strauss
Summary: Making 3 complete seasonal inspection rounds (feb/mar; may/jun; aug/sep) in the same year, ideally carried out by one person, in the forest target area using an orienteering map or a map of similar quality (in paper or in an electronic device; ideally at a scale of 1: 5 000) with additional means, such as GPS, compass and recording devices, so that every accessible place on the target area is screened for new taxa. Screening in a first step for new taxa along all accessible ways in the target area, recording these by determining them directly or later (photo and/or plant material), recording localisation of them by GPS (and optionally by sketching in the orienteering map) and noting optionally their abundance and state. The maximum distance from the way for recording can be e.g. 3 m for herbaceous plants, 10 m for at least 1 m high shrubs and 20 m for at least 2 m high trees and including all taxa which can clearly be recognized from the way. Sketching in the inspection track in the orienteering map. Screening in a second step for new taxa by meandering in 5-20 m broad loops in the accessible subareas between all ways (not including taxa already recorded by screening along ways) and other lines visible on the orienteering map and/or in nature, so that every accessible place can be searched for new taxa. Recording of taxa and additional notes are done as given above for taxa along ways.This sampling method can possibly also be applied to complete plant taxa inventories in large forests in other zones using orienteering maps or maps of similar quality.“O-map/way method-taxa”: A sampling method for complete plant taxa inventories in large forests in the moderate/colline zone using orienteering maps or maps of a similar quality By André Strauss, August 29. 2019

Proper citation: André Strauss 2019. “O-map/way method-taxa”: A sampling method for complete plant taxa inventories in large forests in the moderate/colline zone using orienteering maps or maps of a similar quality. protocols.io https://dx.doi.org/10.17504/protocols.io.6xbhfin Copy   

•   Source:

Authors: Gurdon Institute mediak
Summary: Make LB agar medium.

Proper citation: Gurdon Institute mediak 2019. Make LB agar medium. protocols.io https://dx.doi.org/10.17504/protocols.io.5yfg7tn Copy   

•   Source:

Authors: Tjusls China

Proper citation: Tjusls China 2019. 01 PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.4y7gxzn Copy   

•   Source:

Authors: Frank Aylward
Summary: Week 1Introduction to parsing FASTA files.Commands to be entered into the command line are in bold. Here we will be using various base Unix commands such as head, tail, sort, wget, and others. We will also be using the seqkit tool to process FASTA files. The main page for seqkit is here:https://github.com/shenwei356/seqkit

Proper citation: Frank Aylward 2019. 02: Parsing FASTA files. protocols.io https://dx.doi.org/10.17504/protocols.io.z9mf946 Copy   

•   Source:

Authors: Sarah Hessen-Schmidt

Proper citation: Sarah Hessen-Schmidt 2016. Qubit dsDNA HS/BR Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.e7sbhne Copy   

•   Source:

Authors: Yuan Yan, Haihong Liu, Shuiyuan Xiao

Proper citation: Yuan Yan, Haihong Liu, Shuiyuan Xiao 2018. Sexual Orientation and Mental Health among Undergraduates of 10 Universities in Guangzhou, China. protocols.io https://dx.doi.org/10.17504/protocols.io.pcrdiv6 Copy   

•   Source:

Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are positively selected or depleted by incubating the sample with an anti-human APC conjugated antibody, followed by incubation with magnetic anti-APC Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the APC+ cells, do not discard them if those are the cells of interest. Some of the 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 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Human anti-APC Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7x2hpqe Copy   

•   Source:

Authors: Sarah Giuliani
Group: Coleman Lab
Summary: Monitor fluorescence of cyanobacterial cultures over time using a 96-well plate format. 

Proper citation: Sarah Giuliani 2016. Monitoring fluorescence during cyanobacterial physiology/lysis experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.f28bqhw Copy   

•   Source:

Authors: Daniel Núñez, Tamara Matute, Isaac Núñez, Fernan Federici
Group: Laboratorio de Tecnologias Libres
Summary: Este protocolo describe como aislar bacterias mótiles del género Paenibacillus a partir de muestras ambientales. El protocolo no involucra material de laboratorio especializado.Parte del proyecto Comunicaciones Especulativas: datos en https://osf.io/c542q/wiki/home/codigo en https://github.com/interspecifics/ComunicacionesEspeculativasinfo en http://interspecifics.cc/comunicacionesespeculativas/

Proper citation: Daniel Núñez, Tamara Matute, Isaac Núñez, Fernan Federici 2018. Aislamiento de Paenibacillus sp. desde muestras ambientales. protocols.io https://dx.doi.org/10.17504/protocols.io.nwadfae Copy   

•   Source: