Protocols   Select Another Resource Report Type

Authors: Grant Lin, Michelle Monje
Group: Human Cell Atlas Method Development Community
Summary: This is our standard brain tissue dissociation protocol for cell sorting (Human Cell Atlas).

Proper citation: Grant Lin, Michelle Monje 2018. Brain Tissue Dissociation for Cell Sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.nx7dfrn Copy   

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Authors: Je Hyuk Lee, Evan R. Daugharthy, Jonathan Scheiman, Reza Kalhor, Thomas C. Ferrante, Richard Terry, Brian M. Turczyk, Joyce L. Yang, Ho Suk Lee, John Aach, Kun Zhang, and George M. Church
Group: Human Cell Atlas Method Development Community
Summary: RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Proper citation: Je Hyuk Lee, Evan R. Daugharthy, Jonathan Scheiman, Reza Kalhor, Thomas C. Ferrante, Richard Terry, Brian M. Turczyk, Joyce L. Yang, Ho Suk Lee, John Aach, Kun Zhang, and George M. Church 2018. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.mgsc3we Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The first step in any MERFISH experiment is the design of the oligonucleotide probes that will be used to label individual RNA species. In our current implementation of MERFISH, each oligonucleotide encoding probe consists of three basic components as illustrated in Figure 2. The first region is a 30-nt targeting region that is complementary to a portion of the sequence of the RNA to which it is designed to bind. The second region is a set of sequences that are called readout sequences, which were designed to be complementary and hence only bind to MERFISH readout probes and not other nucleic acid in the cell. Finally, the third region is a set of priming regions used in the construction of these probes, which will be discussed in detail in Probe Construction. In addition to the nucleotide sequences for each of these components, a codebook —the specific set of binary barcodes that will be used and their association with different RNA species of interest—must also be designed. In this section, we provide protocols to design these sequences and to build a codebook. Example code to perform these steps can be found at http://zhuang.harvard.edu/merfish/ .

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Design of Oligonucleotide Probes. protocols.io https://dx.doi.org/10.17504/protocols.io.menc3de Copy   

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Authors: Mandy Xu, Troy Ketela
Group: Human Cell Atlas Method Development Community, CZI START Project
Summary: This protocol has been developed by the Princess Margaret Genomics Centre specifically for Nuc-Seq, both at the bulk and single-cell levels.

Proper citation: Mandy Xu, Troy Ketela 2019. Nuclei-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.8b8hsrw Copy   

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Authors: Marlon Stoeckius, Peter Smibert
Group: Human Cell Atlas Method Development Community
Summary: This protocol is for performing CITE-seq and Cell Hashing in parallel. CITE-seq: 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.Cell Hashing:Sample multiplexing and super-loading on single cell RNA-sequencing platforms.Cell Hashing uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples.

Proper citation: Marlon Stoeckius, Peter Smibert 2018. CITE-seq and Cell Hashing. protocols.io https://dx.doi.org/10.17504/protocols.io.nhqdb5w Copy   

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Authors: Peter Sims, Jinzhou Yuan, Yim L. Cheng
Group: Human Cell Atlas Method Development Community
Summary: (On-chip reverse transcription version, Sims Lab – Jinzhou Yuan and Yim L. Cheng) 7/6/2017

Proper citation: Peter Sims, Jinzhou Yuan, Yim L. Cheng 2019. Microwell-based Single-Cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.yqmfvu6 Copy   

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Authors: Nan Liu
Group: Human Cell Atlas Method Development Community
Summary: Choose your protocol:For Henikoff lab protocol, please see their Nature Protocol paper.Why?The difference between NEB stem loop adaptors and illumina adaptors are their lengths. When ligated to both ends of DNA, they add 64bp and 124bp respectively. This makes huge difference when you do beads based cleaning up.What is this protocol?This protocol is based on library preparation manual of “NEBNext® Ultra™ II DNA Library Prep Kit for Illumina”, NEB E7645, and is specifically modified to make libraries from small DNA fragments (25-70bp). We have successfully applied this protocol to make libraries for CUT&RUN experiments and showed that it greatly increased the preservation of small fragments (Liu, Cell 2018. Fig. S2B [pdf])

Proper citation: Nan Liu 2019. Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645). protocols.io https://dx.doi.org/10.17504/protocols.io.wvgfe3w Copy   

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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 2019. Adult Mouse Spleen Dissociation (On ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ydwfs7e 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: For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. Here, we describe a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second “template-switch” primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.

Proper citation: John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev 2018. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.pbhdij6 Copy   

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Authors: Quy Nguyen
Group: Human Cell Atlas Method Development Community

Proper citation: Quy Nguyen 2018. Human Breast Tissues Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.twbepan Copy   

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

Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Pancreas Biopsy Collection, ex vivo. protocols.io https://dx.doi.org/10.17504/protocols.io.siwecfe Copy   

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Authors: Steven Henikoff, Jorja Henikoff, Hatice Kaya-Okur, Kami Ahmad
Group: Human Cell Atlas Method Development Community
Summary: CUT&Tag@direct uses a modification of Bench-top CUT&Tag which includes incubation in 0.1% SDS post-tagmentation for quantitative release of targeted fragments, followed directly by PCR with Triton-X100 to neutralize the SDS. This protocol is performed in single PCR tubes from nuclei to sequencing-ready libraries and should be suitable for high throughput. The protocol has been enhanced by the addition of hyperaccessibility mapping by Cleavage Under Targeted Accessible Chromatin (CUTAC), where H3K4me2 CUT&Tag samples are tagmented in low salt for mapping of the hyperaccessible site close to the H3K4me2-labeled nucleosomes.In situ tethering for CUT&Tag chromatin profiling.a) The steps in CUT&Tag. Added antibody (green) binds to the target chromatin protein (blue) between nucleosomes (gray ovals) in the genome, and the excess is washed away. A second antibody (orange) is added and enhances tethering of pA-Tn5 transposome (gray boxes) at antibody-bound sites. After washing away excess transposome, addition of Mg++ activates the transposome and integrates adapters (red) at chromatin protein binding sites. After DNA purification genomic fragments with adapters at both ends are enriched by PCR. b) CUT&Tag is performed on a solid support. Unfixed cells or nuclei (blue) are permeabilized and mixed with antibody to a target chromatin protein. After addition and binding of cells to Concanavalin A-coated magnetic beads (M), all further steps are performed in the same reaction tube with magnetic capture between washes and incubations, including pA-Tn5 tethering, integration, and DNA purification.

Proper citation: Steven Henikoff, Jorja Henikoff, Hatice Kaya-Okur, Kami Ahmad 2020. CUT&Tag-direct with CUTAC. protocols.io https://dx.doi.org/10.17504/protocols.io.bmbfk2jn Copy   

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Authors: Carly Martin, 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, Ashley Feirrera, Evan Macosko 2019. Frozen Tissue Nuclei Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.2frgbm6 Copy   

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Authors: Luciano Martelotto
Group: Human Cell Atlas Method Development Community, Single Cell Core, Harvard Medical School
Summary: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al.This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies.The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other samples types.Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.

Proper citation: Luciano Martelotto 2020. 'Frankenstein’ protocol for nuclei isolation from FRESH and FROZEN tissue for snRNA-Seq (10x Genomics Platform). protocols.io https://dx.doi.org/10.17504/protocols.io.bq27myhn Copy   

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Authors: Xuesen Cheng, Yumei Li, Rui Chen
Group: Human Cell Atlas Method Development Community

Proper citation: Xuesen Cheng, Yumei Li, Rui Chen 2020. Nuclei Isolation for SnRNA-seq and SnATAC-seq from Frozen Fresh Human Retina Sample. protocols.io https:// Copy   

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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 human whole blood for scRNAseq analysis.

Proper citation: Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber 2020. COVID Blood Processing for scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bjm6kk9e Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This procedure is used to dissociate adult (10 wk.) mouse pancreas into single cells. The procedure is carried out on ice in order to maintain a more authentic gene expression profile. It is a two-layered dissociation, with each layer consisting of 5 mg/mL type 4 collagenase. The yield is 4400 cells/mg with 94% viability. 

Proper citation: Andrew Potter 2018. Adult mouse pancreas cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.r8rd9v6 Copy   

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

Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Islet Purification - COBE setup. protocols.io https://dx.doi.org/10.17504/protocols.io.sibecan Copy   

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