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Authors: Derek Janssens, Steven Henikoff
Group: protocols.io news, Human Cell Atlas Method Development Community
Summary: The CUT&Tag method is based on antibody tethering of the Tn5 transposase to profile the genome-wide occupancy of DNA-binding proteins, histone modifications and chromatin modifying proteins in situ starting from relatively low cell numbers (1-100K cells). Activation of tethered transposase integrates library adapters at the sites of bound chromatin factors. By taking advantage of protocol modifications that enable Tn5 release and PCR in the same sample reaction chamber, we adapted CUT&Tag to an automated 96 well format. We provide programs and equipment specifications for performing AutoCUT&Tag on a Beckman Coulter Biomek FX liquid handling robot equipped for magnetic separation and temperature control, but in principle the method could be easily translated to other liquid handling units. Using this automated protocol a single operator is able to generate up to 96 CUT&Tag libraries in a single day that are ready to be pooled and sequenced.

Proper citation: Derek Janssens, Steven Henikoff 2020. AutoCUT&Tag: streamlined genome-wide profiling of chromatin proteins on a liquid handling robot. protocols.io https://dx.doi.org/10.17504/protocols.io.bgztjx6n Copy   

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Authors: Roser Vento-Tormo, Regina Hoo
Group: Human Cell Atlas Method Development Community, Vento-Tormo
Summary: This protocol describe the tissue dissociation procedures from human endometrium and pregnancy endometrium samples. This protocol is adapted from Vento-Tormo et al. 2018 Nature with some modification from Prof. Ashley Moffett (Department of Pathology, University of Cambridge).

Proper citation: Roser Vento-Tormo, Regina Hoo 2019. Endometrium dissociation with collagenase. protocols.io https://dx.doi.org/10.17504/protocols.io.76thren 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: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 (25-150bp). 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 2021. 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.bagaibse Copy   

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Authors: Ilias Angelidis, Maximilian Strunz, Herbert Schiller
Group: Human Cell Atlas Method Development Community

Proper citation: Ilias Angelidis, Maximilian Strunz, Herbert Schiller 2019. Cell dissociation of fresh human lung tissue for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.zp2f5qe Copy   

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Authors: Harikrishna Nakshatri
Group: Human Cell Atlas Method Development Community
Summary: This protocol details the cryopreservation of tissues used for primary cell cultures and single cell sequencing.

Proper citation: Harikrishna Nakshatri 2020. Cryopreservation of tissues for primary cell culture and single cell sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bhvjj64n Copy   

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Authors: Kimberly Siletti, Sten Linnarsson
Group: Human Cell Atlas Method Development Community, BICCN
Summary: This protocol describes our preparation of nuclei for single-nucleus RNA sequencing on the 10X Genomics Chromium system, starting from FAC-sorted frozen nuclei. 

Proper citation: Kimberly Siletti, Sten Linnarsson 2019. Preparation of frozen nuclei for single-nucleus RNA seq. protocols.io https://dx.doi.org/10.17504/protocols.io.nx3dfqn Copy   

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Authors: Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: These are Expansion FISH (ExFISH) protocols for super-resolution imaging of RNA structure and location with diffraction-limited microscopes.Please select the appropriate protocol depending on your sample (cells or tissue).

Proper citation: Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden 2018. ExFISH Protocols For Cells and Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.n7bdhin Copy   

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Authors: Gloria Pryhuber, Ravi Misra, Tiru Rangasamy, Thomas Mariani, Gautam Bandyopadhyay, Lisa Rogers, Amanda Howell, Claire Wyman, Heidie Huyck, Siva Solleti
Group: Human Cell Atlas Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)702.B.3 Combined SOP and Worksheet HTC_Lung_Tissue_Digestion_0100616rev061220Purpose and Scope of the Procedure or Laboratory Assay1.To dissociate and isolate single cell suspensions from human lung samples to meet the standards and needs of the Human Tissue Core. .justify:after { content: ""; display:inline-block; width: 100%; } 2.This protocol covers manual gross dissociation of pieces of lung tissue (lobes or cubes) followed by enzymatic digestion and freezer storage as mixed populations of filtered and counted cells for which viability has been determined. .justify:after { content: ""; display:inline-block; width: 100%; } 3.A separate protocol details the technique of freezing, thawing and further identifying and sorting these mixed cell populations. .justify:after { content: ""; display:inline-block; width: 100%; } Scientific Principles or Validation of Procedure .justify:after { content: ""; display:inline-block; width: 100%; } 1.Fujino et al. Am J Respir Cell Mol Biol. 2012 Apr;46(4):422-30 .justify:after { content: ""; display:inline-block; width: 100%; } 2.Barkauskas, C. E., Cronce, M. J., Rackley, C. R., Bowie, E. J., Keene, D. R., Stripp, B. R., ... & Hogan, B. L. (2013). Type 2 alveolar cells are stem cells in adult lung. The Journal of clinical investigation, 123(7), 3025. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Gloria Pryhuber, Ravi Misra, Tiru Rangasamy, Thomas Mariani, Gautam Bandyopadhyay, Lisa Rogers, Amanda Howell, Claire Wyman, Heidie Huyck, Siva Solleti 2020. 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820. protocols.io https://dx.doi.org/10.17504/protocols.io.biz5kf86 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. Mouse P1 Kidney Cold-Active Protease Single Cell Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.q7ddzi6 Copy   

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Authors: Marlon Stoeckius, Peter Smibert
Group: Human Cell Atlas Method Development Community
Summary: This collection contains our main protocols for performing CITE-seq and Cell Hashing, specifically on Drop-seq or 10x Genomics single cell 3′ v2 chemistry. 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 Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.ngzdbx6 Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol was used to generate a single cell suspension from adult human lung tissue. The procedure is carried out on ice, reducing artifact gene expression changes. 

Proper citation: Andrew Potter 2018. Adult human lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rcad2se Copy   

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Authors: 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 (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that prevents premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over ofE. coliDNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. 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: 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.n6wdhfe 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. 6: Protocol optimization for SABER-FISH in tissues. protocols.io https:// Copy   

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Authors: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris
Group: Human Cell Atlas Method Development Community, Morris Lab
Summary: Single-cell technologies are offering unprecedented insight into complex biology, revealing the behavior of rare cell populations that are typically masked in bulk population analyses. One current limitation of single-cell approaches is that lineage relationships are lost as a result of cell processing, restricting interpretations of the data collected. Elegant computational approaches have been developed in an effort to infer these missing observations, but it remains a challenge to reconstruct true reprogramming trajectories using these tools. Although sophisticated lineage tracing solutions to connect cell history with fate are emerging, these protocols are either not compatible with high-throughput scRNA-seq, or require genome editing strategies that are not readily deployed in some systems. Here, our protocol describes a single-cell resolution clonal tracking approach, ‘CellTagging’, based on combinatorial cell indexing, permitting the parallel capture of lineage information and cell identity. CellTagging integrates with high-throughput single-cell RNA-sequencing, where iterative rounds of cell labeling enable the construction of multi-level lineage trees. This straightforward lentiviral-labeling approach can be applied to an array of cell biological applications to simultaneously profile lineage and identity, at single-cell resolution.

Proper citation: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris 2019. Single-cell mapping of lineage and identity via CellTagging. protocols.io https://dx.doi.org/10.17504/protocols.io.yxifxke Copy   

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Authors: Woong-Yang Park, Jay Shin, Shyam Prabhakar
Group: Human Cell Atlas Method Development Community

Proper citation: Woong-Yang Park, Jay Shin, Shyam Prabhakar 2020. Demuxlet Cell Preparation Protocol. protocols.io https:// Copy   

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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 2019. Adult mouse skin dissociation protocol (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ydzfs76 Copy   

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Authors: Brandon Wesley
Group: Human Cell Atlas Method Development Community

Proper citation: Brandon Wesley 2018. Primary Human Liver Dissociation for scSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.nkjdcun 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 Isolation Media Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.sfyebpw Copy   

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Authors: Hatice Kaya-Okur, Steven Henikoff
Group: Human Cell Atlas Method Development Community
Summary: Here we describe a bench top protocol for Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a proteinA-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in one day. We have demonstrated the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells. Version 3 includes an optional light formaldehyde treatment of cells or nuclei prior to antibody addition. Light fixation reduces the tendency of cells or nuclei to clump, which can also be reduced for fixed and unfixed nuclei by omitting digitonin from all steps.

Proper citation: Hatice Kaya-Okur, Steven Henikoff 2020. Bench top CUT&Tag. protocols.io https://dx.doi.org/10.17504/protocols.io.bcuhiwt6 Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for adult (8-10 week) mouse kidney dissociation.

Proper citation: Andrew Potter 2018. Adult mouse kidney dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nf9dbr6 Copy   

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