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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression. 

Proper citation: Andrew Potter 2018. Adult human small intestine cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7cdziw Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes requires a very large number of unique oligonucleotide sequences. The sheer number of unique sequences makes traditional solid phase oligonucleotide synthesis prohibitively expensive in most cases. For example, assuming a modest cost of $0.10/base, an encoding probe length of ~100 nt, and the need for ~50,000 unique oligos for a single MERFISH measurement, the cost of the needed oligos would be $500,000! To circumvent this astronomical cost, we have developed a high-throughput approach to generating these probes which utilizes array-derived synthesis of complex oligonucleotide pools. These arrayderived complex oligonucleotide pools that contain ~100,000 custom designed sequences can be purchased for only a few thousand dollars. The challenge to using these pools is that each individual sequence is provided in quantities far too small to be used directly for labeling. Thus, we developed an enzymatic amplification protocol to generate the encoding probes in high quantity sufficient for RNA FISH experiments using the array-derived complex oligonucleotide pools as templates.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Probe Construction. protocols.io https://dx.doi.org/10.17504/protocols.io.meqc3dw Copy   

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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.qnjdvcn Copy   

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Authors: Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy
Group: Human Cell Atlas Method Development Community

Proper citation: Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy 2018. Human Kidney / Tumour Tissue Disaggregation for Single Cell RNA Sequencing (10x Genomics platform). protocols.io https://dx.doi.org/10.17504/protocols.io.mjyc4pw Copy   

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Authors: Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: DRAFTPurpose:The purpose of this protocol is to outline proper procedures for fixed specimen processing using an automatic paraffin processor and sectioning and staining for histopathological review of normalcy. Scope:This protocol applies to human pancreas specimens that will be processed to paraffin blocks for HuBMAP Tissue Mapping Centers.Expected Outcome:Pancreas samples are processed properly and efficiently to whole slide images for histopathology review. Nuclei should stain blue. Cytoplasm of acinar cells should stain pink with blue zymogen granules. The large blood vessels should demonstrate clear differentiation of RBCs when present, surrounding collagen, and endothelial nuclei with scant cytoplasm.

Proper citation: Martha Campbell Thompson 2021. Human Pancreas Histopathology Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.br7tm9nn Copy   

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Authors: Sonya MacParland, Xue-Zhong Ma, Justin Manuel, Jeff Liu, Gary Bader, Ian McGilvray
Group: Human Cell Atlas Method Development Community
Summary: One of the challenges to describing the human liver on a single cell level is the process of cell dissociation. Cell dissociation methods can be quite damaging to the cells and can lead to a biasing of the transcriptome. We have developed gentle surgical and perfusion techniques to isolate and transcriptionally profile viable resident cells from liver samples taken from deceased donor liver grafts prior to organ transplantation. 

Proper citation: Sonya MacParland, Xue-Zhong Ma, Justin Manuel, Jeff Liu, Gary Bader, Ian McGilvray 2018. Human Liver Caudate Lobe Dissociation for ScRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.m9sc96e Copy   

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

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

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Authors: Jia Ren Lin, Benjamin Izar, Shaolin Mei, Shu Wang, Peter Sorger
Group: Human Cell Atlas Method Development Community, Laboratory of Systems Pharmacology, Cyclic Immunofluorescence
Summary: The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases.  t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

Proper citation: Jia Ren Lin, Benjamin Izar, Shaolin Mei, Shu Wang, Peter Sorger 2018. tCyCIF protcol (Leica BOND) version 0.1. protocols.io https://dx.doi.org/10.17504/protocols.io.rpxd5pn Copy   

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Authors: Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy
Group: Human Cell Atlas Method Development Community

Proper citation: Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy 2018. Human Kidney / Tumour Tissue Disaggregation for Fluorescence-activated cell sorting (bulk or single cell) and flow cytometeric analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ntfdejn Copy   

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Authors: Derek Janssens, Steven Henikoff
Group: Human Cell Atlas Method Development Community
Summary: We previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein. Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. In the manuscript attached to this version of the protocol, Meers et al. introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility; 2) a modified digestion protocol that prevents premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. Here we provide an updated CUT&RUN protocol that incorporates these enhancements, and provides three different options for the CUT&RUN MNase digestion reaction that are helpful to improve data quality or to increase throughput. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

Proper citation: Derek Janssens, Steven Henikoff 2019. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io https://dx.doi.org/10.17504/protocols.io.zcpf2vn Copy   

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Authors: Ran Zhou, Oni Basu
Group: Human Cell Atlas Method Development Community, Helmsley project_Basu lab
Summary: Isolating high-quality nuclei from intestinal surgical tissues is critical for single cell RNA/ATAC-seq. This protocol provides details on nuclei preparation for such application, with an overall working time of less than an hour, if FACS sorting is not incorporated.

Proper citation: Ran Zhou, Oni Basu 2020. Nuclei prep for single cell RNA/ATAC seq from intestinal surgical samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bmbak2ie Copy   

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Authors: Steven Henikoff, Derek Janssens, Hatice Kaya-Okur, Jorja Henikoff, Kami Ahmad
Group: Human Cell Atlas Method Development Community
Summary: CUT&Tag@home 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.

Proper citation: Steven Henikoff, Derek Janssens, Hatice Kaya-Okur, Jorja Henikoff, Kami Ahmad 2020. CUT&Tag@home. protocols.io https://dx.doi.org/10.17504/protocols.io.bd26i8he Copy   

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Authors: Emi Sei, Shanshan Bai, Nicholas Navin
Group: Human Cell Atlas Method Development Community
Summary: This protocol can be used to prepare nuclear suspensions from fresh or frozen tissue for single cell DNA or RNA sequencing experiments. For RNA analysis it is recommended to use fresh tissue samples, due to RNA degradation that occurs during freeze- thaw cycles, however for DNA analysis either frozen or fresh tissue can be used. The protocol has been tested on breast tissues, and many frozen tumor tissues including breast, colon prostate and pancreatic samples.

Proper citation: Emi Sei, Shanshan Bai, Nicholas Navin 2018. Dissociation of Nuclear Suspensions from Human Breast Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.t3aeqie 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. Sampling of Human Islets for Quality Control Purposes. protocols.io https://dx.doi.org/10.17504/protocols.io.sizecf6 Copy   

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Authors: Yang Sun, George Hartoularos
Group: Human Cell Atlas Method Development Community
Summary: This protocol is for staining with AbSeq and multiplexing 16 different samples from distinct individuals into the same 10x run.

Proper citation: Yang Sun, George Hartoularos 2018. Muxab-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.vdme246 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. SABER-FISH – Signal amplification for multiplexed fluorescence in situ hybridization assays. protocols.io https:// Copy   

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Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The basic decoding of MERFISH data—the identification of specific RNA species for each spot in the sample—consists of three basic steps. First, fluorescent spots must be identified in all images. Second, slight changes in the stage position between images of the same region from different hybridization rounds must be corrected. Finally, sets of fluorescent spots that occupy the same location in the sample must be decoded into binary barcodes, which, in turn, are decoded into the specific RNA species. After these RNAs have been decoded, there are a series of simple calculations that can often be performed on the measured barcodes to determine important parameters associated with the performance of the measurements, such as estimates of the binary errors made at each bit or the relative confidence that can be ascribed to the counts for each RNA species. In this section, we provide protocols for performing these basic computational tasks. Example software and data can be found at: http://zhuang.harvard.edu/merfish/.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Data Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.metc3en Copy   

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Authors: Yun Renee Zhang, Brian Aevermann, Richard Scheuermann
Group: Human Cell Atlas Method Development Community
Summary: FR-Match is a supervised cell phenotype matching strategy for cluster-to-cluster cell transcriptome integration across scRNAseq experiments. An R package and Shiny application are provided at https://github.com/JCVenterInstitute/FRmatch.

Proper citation: Yun Renee Zhang, Brian Aevermann, Richard Scheuermann 2020. FR-Match: cell type matching for scRNAseq data. protocols.io https://dx.doi.org/10.17504/protocols.io.bmyfk7tn Copy   

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Authors: Banushree Kumar, Simon Elsässer
Group: Human Cell Atlas Method Development Community
Summary: The need for quantitative ChIP-seq has been widely appreciated, and a number of quantitative methods have been proposed. Mint-ChIP, developed by the Bernstein lab provides a streamlined one-pot chromatin barcoding and a post ChIP linear amplification that requires only one adaptor per chromatin fragment. Here, we introduce unique molecule (unique molecular identifier [UMI]) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTEChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. For paired-end mapping, we modified the linear amplification strategy introduced in Mint-ChIP by priming the cDNA synthesis from a 30 adaptor (RA3) ligated onto the amplified RNA, thus maintaining the original genomic fragment length, retrieving atypical mononucleosomal fragment length for histone ChIP. We have also optimized the ligation conditions and stoichiometry of adaptors to chromatin to enable sequencing of the input. We show that MINUTE-ChIP is a sensitive method with a large linear dynamic range for accurately quantifying relative differences in genome-wide histone modification patterns across multiple pooled samples.

Proper citation: Banushree Kumar, Simon Elsässer 2019. MINUTE ChIP. protocols.io https://dx.doi.org/10.17504/protocols.io.8nkhvcw Copy   

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Authors: Luca Tosti, Christian Conrad
Group: Human Cell Atlas Method Development Community
Summary: A protocol to isolate nuclei from snap-frozen pancreatic tissue and protect RNA from RNAse-mediated degradation

Proper citation: Luca Tosti, Christian Conrad 2020. Nuclei isolation from snap frozen human pancreatic tissue using a citric acid buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.tpxempn Copy   

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