Protocols   Select Another Resource Report Type

Authors: David Kotol
Group: Human Cell Atlas Method Development Community, Human Protein Atlas
Summary: Here, a workflow for sample preparation and quantification of brain and pancreatic tissue proteins with use of heavy labelled protein standards (QPrESTs) is described. The standards are added before the digestion ensuring high reproducibility and elimination of nuances apearing during proteolytical cleavage of proteins and further steps such as solid phase extraction. QPrESTs are heavy labelled recombinant protein fragments covering regions of proteins with low homology and span over 50-150 aminoacids. They were produced as a part of Human Protein Atlas project in auxotrofic E.coli and individually purified, serving as an excelent resource for MS-based protein quantification.

Proper citation: David Kotol 2019. Tissue lysis and digestion for MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yguftww Copy   

•   Source:

Authors: Arnav Moudgil, Robi D. Mitra
Group: Human Cell Atlas Method Development Community, Transposon Calling Cards
Summary: This protocol describes how to create calling card libraries from single cell RNA. We assume you have successfully transformed cells with piggyBac self-reporting transposons and either undirected piggyBac transposase or your favorite transcription factor (YFTF) fused to piggyBac. We also assume you have optimized the dissociation protocol for your specific cells or tissues and can generate single cell suspensions.

Proper citation: Arnav Moudgil, Robi D. Mitra 2019. Single Cell Calling Cards Library Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.xwifpce Copy   

•   Source:

Authors: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin
Group: Human Cell Atlas Method Development Community

Proper citation: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. 4: User-friendly protocol: Cost-efficient Primer Exchange Reaction (PER) concatemerization (SABER-FISH). protocols.io https:// Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol used to dissociate adult (8-10 wk) mouse spleen into single cells. Attained >95% viability, a variety of cell sizes, and ~10 million cells from 12 mg tissue.

Proper citation: Andrew Potter 2018. Adult Mouse Spleen Dissociation (On ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q5cdy2w Copy   

•   Source:

Authors: Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko
Group: Human Cell Atlas Method Development Community
Summary: Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab.

Proper citation: Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko 2019. Frozen Tissue Nuclei Extraction (v2). protocols.io https://dx.doi.org/10.17504/protocols.io.7xchpiw Copy   

•   Source:

Authors: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol describes the isolation of lymphocytes and pan-mononuclear cells from airway samples.

Proper citation: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber 2020. COVID Airway Processing for scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bjj8kkrw Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol was developed to dissociate adult (8-10 wk) mouse skin "on ice". It utilizes two layers of digestion with a Bacillus licheniformis protease cocktail, combined with mechanical disruption from a dounce homogenizer. The cell yield is 3000 cells/mg.

Proper citation: Andrew Potter 2018. Adult mouse skin dissociation protocol (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.tupenvn Copy   

•   Source:

Authors: Heather Eckart, Ran Zhou, Nadia Khan
Group: Human Cell Atlas Method Development Community, Helmsley project_Basu lab
Summary: This protocol provides a procedure for human intestinal organoid dissociation into a single cell suspension and nuclei isolation prior to Single Cell ATAC-Sequencing.

Proper citation: Heather Eckart, Ran Zhou, Nadia Khan 2020. Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bmdbk22n Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol is used to dissociate adult (8-10 wk) mouse ear "on ice" (in order to reduce gene expression artifacts). The cell yield is 9000 cells/mg (213000 cells total from 23 mg tissue), with 98% viable. The protocol involves a 2-layered dissociation, incubating on ice with bacillus licheniformis enzyme. The dissociation is augmented by the use of a dounce homogenizer to help break up the tissue.

Proper citation: Andrew Potter 2018. Adult mouse ear dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ucwesxe Copy   

•   Source:

Authors: Adam Hunter
Group: Human Cell Atlas Method Development Community

Proper citation: Adam Hunter 2018. CGAP MACS Live Dead Separation. protocols.io https://dx.doi.org/10.17504/protocols.io.qz5dx86 Copy   

•   Source:

Authors: Anna Wilbrey-Clark, Adam Hunter
Group: Human Cell Atlas Method Development Community

Proper citation: Anna Wilbrey-Clark, Adam Hunter 2019. CGAP Human Lung Dissociation - Tissue Stability Study. protocols.io https://dx.doi.org/10.17504/protocols.io.34kgquw Copy   

•   Source:

Authors: Junyue Cao, Jay Shendure
Group: Human Cell Atlas Method Development Community
Summary: Single-cell combinatorial indexing (‘sci-’) is a methodological framework that employs split-pool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. Although the throughput of ‘sci-’ methods increases exponentially with the number of rounds of indexing, this potential has yet to be fully realized because of other factors such as the rate of cell loss and the limited reaction efficiency of some steps. To address this, we developed and extensively optimized 3-level sci-RNA-seq (sci-RNA-seq3), resulting in a workflow that can profile millions of cells per experiment. As previously multiple samples (e.g. replicates, timepoints, etc.) can be barcoded during the first round of indexing and concurrently processed.

Proper citation: Junyue Cao, Jay Shendure 2020. sci-RNA-seq3. protocols.io https://dx.doi.org/10.17504/protocols.io.9yih7ue Copy   

•   Source:

Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.smFISH is a powerful technique because it allows the quantitative measurement of the exact copy number and spatial distribution of individual mRNAs within single cells in intact tissues. MERFISH extends these powerful abilities to the transcriptome scale by massively multiplexing smFISH measurements by encoding individual RNA species with error-robust barcodes and reading out the barcodes via a series of smFISH measurements. We anticipate this method to be relatively easily adopted by other laboratories and the techniques described in this collection should facilitate these efforts as will the supporting protocols and code that can be found at: http://zhuang.harvard.edu/merfish/.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.mepc3dn Copy   

•   Source:

Authors: Brian Hie, Bryan Bryson, Bonnie Berger
Group: Human Cell Atlas Method Development Community
Summary: Scanorama enables efficient integration of heterogeneous scRNA-seq data sets.

Proper citation: Brian Hie, Bryan Bryson, Bonnie Berger 2019. Scanorama. protocols.io https://dx.doi.org/10.17504/protocols.io.9gch3sw Copy   

•   Source:

Authors: Benjamin Emert
Group: Human Cell Atlas Method Development Community, RajLab
Summary: Protocol for making invertedClampFISH probes. 

Proper citation: Benjamin Emert 2018. invertedClampFISH ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.qnkdvcw Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for adult (8-10 week) mouse kidney dissociation performed on ice to reduce artifact gene expression. The first layer, consisting of collagenase digestion, breaks down the tissue and releases some cells and glomeruli and tubules. The second layer consists of bacillus licheniformis digestion for 15 min. augmented with a thermomixer at 1400 RPM and passaging with a 27 gauge needle. The second layer is meant to thoroughly break up remaining tubules and glomeruli, releasing cells such as podocytes. The final yield is 250K cells from 18 mg tissue with 98% viability, approximately 14,000 cells/mg tissue. Approximately 1% of released cells are podocytes (visualized using kidneys from MAFB-GFP+ mice using a hemocytometer).

Proper citation: Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rnmd5c6 Copy   

•   Source:

Authors: Jafar S Jabbari, Luyi Tian
Group: Human Cell Atlas Method Development Community
Summary: This protocol describes the subsampling of 10x Chromium generated single cell GEMs after reverse transcription for cDNA amplification. The protocol enables transcriptome sequencing of full-length cDNA from a flexible number of single cells captured on Chromium using long-read sequencers such as Nanopore and PacBio, in addition to preparing short-read Illumina libraries. The protocol is compatible with 10x Single Cell 3' and 5' Gene Expression kits including feature barcoding and also other 10x compatible multiomics methodologies such as CITE-seq.

Proper citation: Jafar S Jabbari, Luyi Tian 2019. Massively parallel long-read sequencing of single cell RNA isoforms. protocols.io https://dx.doi.org/10.17504/protocols.io.8d9hs96 Copy   

•   Source:

Authors: Kenny Roberts, Liz Tuck
Group: Human Cell Atlas Method Development Community
Summary: For many cutting-edge spatial transcriptomic analysis methods, it is essential to prepare and store tissue in a manner that preserves RNA integrity, tissue morphology, and spatial orientation.This protocol describes the process of freezing fresh or fixed and cryoprotected tissues in optimal cutting temperature compound (OCT) using an isopentane-dry ice slurry. Rapid controlled freezing preserves RNA and mimises ice damage, while embedding in OCT prepares tissues for future cryosectioning.

Proper citation: Kenny Roberts, Liz Tuck 2019. Embedding and freezing fresh human tissue in OCT using isopentane. protocols.io https://dx.doi.org/10.17504/protocols.io.95mh846 Copy   

•   Source:

Authors: Lance Peter, Mei-I Chung, Nicholas E. Banovich
Group: Human Cell Atlas Method Development Community

Proper citation: Lance Peter, Mei-I Chung, Nicholas E. Banovich 2019. Single Cell Dissociation of Fresh Lung Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.7xkhpkw Copy   

•   Source:

Authors: Ran Zhou
Group: Human Cell Atlas Method Development Community, Helmsley project_Basu lab
Summary: The protocol is adapted from Fujii's and Smillies's reports for single cell transcriptome analysis from human intestines. It provides details on acquirement of single cell suspension from epithelium and lamina propria. This methods is modified to generate appropriate meterials from patient's intestinal biopsies for sinlge-cell transcriptome and genomic applications.

Proper citation: Ran Zhou 2020. Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications. protocols.io https://dx.doi.org/10.17504/protocols.io.bde3i3gn Copy   

•   Source: