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Authors: Roser Vento-Tormo
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol details the steps and methods of PBMC purification

Proper citation: Roser Vento-Tormo 2020. Purification of PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.bjinkkde Copy   

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Authors: Shanshan Bai, Emi Sei, Nicholas E. Navin
Group: Human Cell Atlas Method Development Community
Summary: This protocol was developed for the human breast cell atlas (HBCA) project to obtain high-viability cell suspensions from freshly dissociated breast tissues from human patients. There are two options for performing this protocol: rapid-dissociation (15-30 min) or exhaustive dissociation (overnight). In our experience the rapid dissociation protocol will provide higher cell viability and better representation of breast cell types, however the total number of cells dissociated may be lower after enumeration. In contrast the exhaustive protocol provides higher cell numbers and maintains good cell viability but may result in skewing of cell type representations over the long time period. The rapid-dissociation protocol was used to generate most of the cell suspensions for the MD Anderson human breast cell atlas project and has been optimized and tested more extensively.

Proper citation: Shanshan Bai, Emi Sei, Nicholas E. Navin 2018. Dissociation of Single Cell Suspensions from Human Breast Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.t3geqjw Copy   

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Authors: Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald
Group: Human Cell Atlas Method Development Community, CIRTNR2FIC

Proper citation: Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald 2019. Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.wztff6n Copy   

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Authors: Derek Janssens, Kami Ahmad, Steven Henikoff
Group: Human Cell Atlas Method Development Community
Summary: CUT&RUN is an antibody-targeted nuclease-cleavage method that profiles the genome-wide occupancy of DNA-binding proteins, histones and chromatin modifying proteins in situ with exceptional sensitivity and resolution. Here we provide a protocol to perform CUT&RUN in an automated 96 well format using a Beckman Biomek FX liquid handling robot equipped for magnetic separation and temperature control. To circumvent the need to purify small amounts of DNA prior to library preparation, we developed a method to polish the DNA ends in chromatin fragments for direct ligation of Illumina library adapters. This allows the samples to be kept in a 96 well format throughout the DNA end-polishing and adapter ligation steps. Deproteinated CUT&RUN libraries are purified on the Biomek using Ampure XP magnetic beads both before and after PCR enrichment. This AutoCUT&RUN protocol allows a single operator to generate up to 96 libraries in two days that are ready to be pooled and sequenced. The easy, cost-effective workflow makes AutoCUT&RUN an attractive tool for high-throughput characterization of chromatin features, and because AutoCUT&RUN generates high-quality profiles from frozen tissue samples this method is also suitable for rapidly profiling the epigenetic content of patient samples.

Proper citation: Derek Janssens, Kami Ahmad, Steven Henikoff 2018. AutoCUT&RUN: genome-wide profiling of chromatin proteins in a 96 well format on a Biomek. protocols.io https://dx.doi.org/10.17504/protocols.io.ufeetje Copy   

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Authors: James Lyon, Aliya Spigelman, Patrick Macdonald, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community, CIRTNR2FIC
Summary: This collection of protocols describe the processes used by the Alberta Diabetes Institute IsletCore for the isolation of human pancreatic islets of Langerhans for research. In addition, protocols related to quality control and the cryopreservation of human islets are included.The ADI IsletCore program is described in full here:Lyon J, Manning Fox JE, Spigelman AF, Kim R, Smith N, O'Gorman D, Kin T, Shapiro AMJ, Rajotte RV, MacDonald PE (2016) Research-focused isolation of human islets from donors with and without diabetes at the Alberta Diabetes Institute IsletCore. Endocrinology, 157(2): 560-569.

Proper citation: James Lyon, Aliya Spigelman, Patrick Macdonald, Jocelyn Manning Fox 2019. ADI IsletCore Protocols for the Isolation, Assessment and Cryopreservation of Human Pancreatic Islets of Langerhans for Research Purposes. protocols.io https://dx.doi.org/10.17504/protocols.io.x3mfqk6 Copy   

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Authors: Jeffrey Koenitzer, Ben Humphreys
Group: Human Cell Atlas Method Development Community
Summary: This protocol is for nuclei isolation from mouse lung for single nucleus RNASeq.It is adapted directly from Joshi et al., with adjustments to RNase inhibitor concentrations and removal of FACS sorting steps.

Proper citation: Jeffrey Koenitzer, Ben Humphreys 2020. Nuclei isolation from mouse lung for single nucleus RNASeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bdv2i68e Copy   

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Authors: Stephanie Lozano
Group: Human Cell Atlas Method Development Community
Summary: Recent advances in single cell mRNA sequencing technology such as Drop-seq (Cell, 2015) have allowed the investigation of cellular composition and processes within complex tissues. Although some fresh tissue samples are available for prospective studies, there is an abundance of archived clinical samples readily available in biobanks to investigate different biological processes. However, archived tissues are not easily amenable to tissue digestion, yielding cells with compromised integrity. An alternate method to single-cell sequencing called DroNc-seq (Nature Methods, 2017) employs the use of single nuclei. DroNc-seq takes advantage of the higher structural integrity of the nuclear membrane as compared to the cellular membrane. This permits exploration of tissues for which a viable single-cell suspension cannot be obtained, such as cardiac tissue. With growing interest in cataloguing cardiac transcriptomic data for the Human Cell Atlas Project, the need for optimized nuclei isolation protocols proves critical. Here, we develop a method for obtaining single-nucleus suspensions from snap-frozen human heart tissue for use in DroNc-seq, expanding the variety of samples from which we can obtain transcriptomic data.

Proper citation: Stephanie Lozano 2018. Human Heart Nuclei Isolation for DroNc-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.vgue3ww Copy   

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Authors: Patrick Macdonald, James Lyon, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community

Proper citation: Patrick Macdonald, James Lyon, Jocelyn Manning Fox 2019. IsletCore Equipment Setup for Human Islet Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.sg4ebyw Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for thymus dissociation (10-week old CD-1 female). 

Proper citation: Andrew Potter 2018. Thymus dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.ngcdbsw Copy   

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Authors: Evan Macosko, Melissa Goldman
Group: Human Cell Atlas Method Development Community
Summary: We have continued to optimize this protocol since doing the experiments in the Macosko et al. Cell paper. What we are sharing here is our current, optimized protocol. As a result, this protocol will not precisely match the methods section of the paper, which is a description of the experiments done in the paper. Please feel free to use these optimizations without author consideration - just acknowledge “helpful advice from Evan Macosko, Melissa Goldman and Steve McCarroll”, and please also include the URL and version number of the protocol in your methods section. This protocol also includes hints, suggestions and images that we could not fit into the the methods section of the paper.

Proper citation: Evan Macosko, Melissa Goldman 2018. Drop-Seq Laboratory Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mkbc4sn Copy   

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Authors: John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev
Group: Human Cell Atlas Method Development Community
Summary: This is a Single-cell RNA-Seq expression analysis Support Protocol for Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. 

Proper citation: John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev 2018. Single-cell RNA-Seq expression analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.pbidike Copy   

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Authors: Abbas Rizvi, Elena Kandror, Tom Maniatis
Group: Human Cell Atlas Method Development Community

Proper citation: Abbas Rizvi, Elena Kandror, Tom Maniatis 2019. Human Spinal Cord Nuclei Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.9yhh7t6 Copy   

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Authors: Stephen Clark
Group: Human Cell Atlas Method Development Community
Summary: Here we describe the full wetlab protocol for scNMT-seq (single-cell nucleosome position, methylome and transcriptome sequencing), a plate-based single-cell multi-omic method.In scNMT-seq, a GpC methyltransferase is used to label non-nucleosome bound DNA thus encoding chromatin accessiblity information within the genomic DNA. Bisulfite conversion is then used to discriminate the two chromatin states which can be read from GpC dinucleotide positions. Endogenous DNA methylation is read from the CpG positions of the same molecules and gene expression data is obtained by capturing poly-A RNA on magnetic beads and pysically separating before the gDNA is bisulfite converted. This mRNA on beads is pocessed using Smart-seq2. The protocol is carried out in 96w plates and typically takes 3-4 days to complete.

Proper citation: Stephen Clark 2019. scNMT-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.3b8girw Copy   

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Authors: Andrew Potter, Steve Potter
Group: Human Cell Atlas Method Development Community
Summary: Method used to derive single cell suspension from P1 mouse kidneys on ice, generating a cell suspension with greatly reduced artifact gene expresion changes and suitable for downstream analysis using 10x Chromium or DropSeq. 

Proper citation: Andrew Potter, Steve Potter 2018. P1 Kidney Cold-Active Protease Single Cell Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nzkdf4w Copy   

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Authors: Marlon Stoeckius
Group: Human Cell Atlas Method Development Community
Summary: This protocol is for performing CITE-seq only. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a multimodal single cell phenotyping method developed in the Technology Innovation lab at the New York Genome Center in collaboration with the Satija lab.CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout.  Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq).This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches.

Proper citation: Marlon Stoeckius 2018. CITE-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.ngydbxw Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The preparation and staining of samples for MERFISH follows closely the typical protocols used for smFISH (Raj et al., 2008). However, there are a few places in which we have modified these protocols to optimize MERFISH staining. Again, RNase contamination can destroy samples, so care should be taken to work in an RNase-free environment.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Sample Preparation and Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.merc3d6 Copy   

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Authors: Linas Mazutis, Ignas Masilionis, Ojasvi Chaudhary
Group: Human Cell Atlas Method Development Community, NCIHTAN

Proper citation: Linas Mazutis, Ignas Masilionis, Ojasvi Chaudhary 2020. Frozen tissue dissociation for single-nucleus RNA-Seq. protocols.io https:// Copy   

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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. 7: User-friendly protocol: Retina Tissue Sections RNA FISH. protocols.io https:// Copy   

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Authors: Ran Zhou, Oni Basu
Group: Human Cell Atlas Method Development Community, Helmsley project_Basu lab
Summary: This protocol provides an efficient method to isolate nuclei from human intestinal biopsy samples for single cell applications (RNA-seq or ATAC-seq).

Proper citation: Ran Zhou, Oni Basu 2020. Nuclei isolation from human intestinal biopsic tissue for single-cell genomic applications. protocols.io https://dx.doi.org/10.17504/protocols.io.bma2k2ge Copy   

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Authors: Anna Wilbrey-Clark, Adam Hunter
Group: Human Cell Atlas Method Development Community
Summary: The aim with this protocol is to pull together what we have learnt from dissociating other tissues, to generate a 'generic' protocol that can be used on multiple tissues for single cell RNA sequencing. To date (June 2019) we have mainly used this on human adult lung and gut and it is definitely still in development! We are also testing replacing liberase TL with liberase DH. The trypsin step may or may not be necessary depending on tissue, but can liberate additional cells compared to the liberase alone.

Proper citation: Anna Wilbrey-Clark, Adam Hunter 2019. Human adult generic tissue dissociation *in development*. protocols.io https://dx.doi.org/10.17504/protocols.io.39ygr7w Copy   

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