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Authors: Daniel Gyllborg, Chika Yokota, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: In situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved tissue. This protocol can be used to detect RNA molecules at the single cell level to aid in the identification of cell types according to their gene expression. The technique uses padlock probes to target desired genes of interest and rolling circle amplification to amplify signal for a high throughput methodolgy of spatial transcriptomics. With the use of barcode sequencing, identification of numerous genes is possible through multiplexing.

Proper citation: Daniel Gyllborg, Chika Yokota, Mats Nilsson 2019. In situ sequencing for RNA analysis in tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.s8vehw6 Copy   

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Authors: Song Chen, Blue B. Lake, Sarah Urata, Kun Zhang
Group: Human Cell Atlas Method Development Community, KPMP
Summary: The protocol presented here is a Drop-Seq protocol modified for single nuclei. The original Drop-Seq protocol comes from the McCarroll Lab in the Department of Genetics, Harvard Medical School.http://mccarrolllab.org/download/905/

Proper citation: Song Chen, Blue B. Lake, Sarah Urata, Kun Zhang 2020. Single Nucleus Drop-seq (snDrop-seq). protocols.io https://dx.doi.org/10.17504/protocols.io.zmvf466 Copy   

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Authors: Luciano Martelotto
Group: Human Cell Atlas Method Development Community
Summary: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas/paragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).

Proper citation: Luciano Martelotto 2020. Protocol for nuclei isolation from fresh and frozen tissues for parallel snRNA-Seq and snATAC-Seq on 10x ChromiumTM platform using the same nuclei preparation (UPDATED version). protocols.io https://dx.doi.org/10.17504/protocols.io.bdeni3de Copy   

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Authors: Adam Hunter
Group: Human Cell Atlas Method Development Community
Summary: Mechanical dissociation of human spleen tissue to single cells for use in 10X chromium scRNA-Seq preparation. 

Proper citation: Adam Hunter 2018. CGAP Human Spleen Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.qz3dx8n 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. This unit describes 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. Curr. Protoc. Mol. Biol. 107:4.22.1‐4.22.17. © 2014 by John Wiley & Sons, Inc.

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.n6gdhbw 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. 2: User-friendly protocol: Oligo ordering and preparation (SABER-FISH). protocols.io https:// Copy   

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Authors: Jongeun Park
Group: Human Cell Atlas Method Development Community
Summary: This is protocol for fetal thymus dissociation. (Can be applied to other fetal tissues)Can be also adapted to adult thymus or lymphoid organs. (Volume of solution needs to be adjusted to take account of larger tissue size, also consider multiple rounds of digestion in this case)

Proper citation: Jongeun Park 2018. Cambridge Fetal Thymus Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.wedfba6 Copy   

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Authors: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole
Group: Human Cell Atlas Method Development Community, Human BioMolecular Atlas Program (HuBMAP) Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Purpose and Scope of the Procedure- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC - Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC

Proper citation: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole 2020. 615.1 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bjtnknme 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 imaging process for MERFISH involves several fluid handling steps, e.g. introduction of hybridization buffers and wash and imaging buffers, in conjunction with the collection of smFISH images and the photobleaching of the sample. While it would be possible to perform these fluid exchange and imaging steps manually, we strongly recommend the use of an integrated and automated fluid exchange and imaging approach. Here we provide protocols for how to construct a fluid handling system that should be compatible with a variety of microscopes. We also discuss the basic protocols for hybridizing readout probes to samples on the microscope, imaging the sample, and bleaching residual signal.

Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.mesc3ee Copy   

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Authors: Brian Aevermann, Richard Scheuermann
Group: Human Cell Atlas Method Development Community
Summary: NS-Forest is an alogrithm that determines the minimum set of genes that are necessary and sufficient to define a cell type cluster derived from single cell RNAseq expression data. Development and stable releases can be found at : https://github.com/JCVenterInstitute/NSForest

Proper citation: Brian Aevermann, Richard Scheuermann 2018. NS-Forest version 2. protocols.io https://dx.doi.org/10.17504/protocols.io.un7evhn 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. 1: User-friendly protocol: Probe set design (SABER-FISH). protocols.io https:// Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol can be used to dissociate adult human kidney “on ice” - maintaining authentic gene expression profiles. It was designed using a mix of Collagenases (Type 4, and A) which provide broad proteolytic activity, but preferentially cleave extracellular bonds, largely leaving cells intact. The total incubation time is 1 hour 20 minutes divided into two layers. At the end of the procedure, RBC lysis is performed. The total yield at the end of the procedure is ~1200 (non-RBC) cells released per mg tissue with 87% viability.In the digest mix, there is trypsin inhibitor from soybean which is designed to limit the activity of tryptic proteins in the collagenase mix which can damage the integrity of the cell. There is also 5 mM CaCl2, an activator of collagenase activity, in addition to DNAse - which chews up DNA released from dead cells, reducing cell clumping. The dissociation itself it carried out in two layers. The first layer is 30 minutes and includes trituration and shaking. After this layer, tissue clumps are settled for 1 min, and the supernatant containing released cells is removed and filtered using a 30 µM filter and rinsed with ice-cold PBS-BSA. This helps to preserve the integrity of released cells while continuing the digest clumps of undissociated cells. To the residual clumps, an additional 1 mL of enzyme mix is added and the digestion is continued for 50 additional minutes (1 hr 20 mins total time).

Proper citation: Andrew Potter 2018. Adult human kidney tissue cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q6cdzaw 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. Adult mouse thymus dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7edzje Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for human intestine cell dissociation. 

Proper citation: Andrew Potter 2018. Intestine cell dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nzjdf4n Copy   

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Authors: Sara H Rouhanifard, Ian A Mellis, Margaret Dunagin, Sareh Bayatpour, Orsolya Symmons, Allison Cote, Arjun Raj
Group: Human Cell Atlas Method Development Community
Summary: Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present click-amplifying FISH (clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and extremely high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that the modular design of clampFISH probes enables multiplexing, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH works in tissue samples.

Proper citation: Sara H Rouhanifard, Ian A Mellis, Margaret Dunagin, Sareh Bayatpour, Orsolya Symmons, Allison Cote, Arjun Raj 2018. ClampFISH . protocols.io https://dx.doi.org/10.17504/protocols.io.qeydtfw Copy   

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Authors: Peter J. Skene, Steven Henikoff
Group: Human Cell Atlas Method Development Community
Summary: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.In summary, CUT&RUN has several advantages over ChIP-seq: (1) The method is performed in situ in non-crosslinked cells and does not require chromatin fragmentation or solubilization; (2) The intrinsically low background allows low sequence depth and identification of low signal genomic features invisible to ChIP; (3) The simple procedure can be completed within a day and is suitable for robotic automation; (4) The method can be used with low cell numbers compared to existing methodologies; (5) A simple spike-in strategy can be used for accurate quantitation of protein-DNA interactions. As such, CUT&RUN represents an attractive replacement for ChIPseq, which is one of the most popular methods in biological research.

Proper citation: Peter J. Skene, Steven Henikoff 2018. 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.mgjc3un Copy   

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Authors: Nikita Joshi, Alexander Misharin
Group: Human Cell Atlas Method Development Community
Summary: We aimed to develop a protocol for isolation of the single nuclei from the archival frozen human lung tissue suitable for single cell RNA-seq using standard 10x Genomics chemistry. The protocol utilizes standard nuclei isolation buffer (Nuclei EZ buffer) supplemented with RNase inhibitor and a protease inhibitor. For the homogenization step, we elected to use C Tube and GentleMACS tissue dissociation as a way to standardize the homogenization procedure. To maintain nuclei integrity we skipped washing steps and instead use large volumes of washing buffer and proceed to FACSorting immediately after lysis for nuclei purification.This protocol produces a good yield of nuclei and diverse libraries, with multiple cell types being detected. While the number of detected genes and UMI per nuclei is lower than for single cell RNAseq, it is sufficient for identification of the specific cell types, including rare populations. Moreover, snRNAseq allowed resolution of the cell types that are intimately integrated into the lung matrix and otherwise hard to resolve: fibroblasts and type 1 alveolar epithelial cells.We thank Oni Basu (UChicago), Luciano Martelotto (Monash University), Nicole Abreu (10x Genomics) and Sharmila Chatterjee (10x Genomics) for their advice.

Proper citation: Nikita Joshi, Alexander Misharin 2019. Single-nucleus isolation from frozen human lung tissue for single-nucleus RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.zu8f6zw Copy   

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Authors: Ran Zhou, Oni Basu
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, Oni Basu 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.bkp6kvre Copy   

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

Proper citation: Adam Hunter 2018. CGAP Human Oesophagus Epithelium Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.qz8dx9w Copy   

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Authors: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to 500 μm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 μm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Proper citation: Elizabeth Butterworth, Wesley Dickerson, Vindhya Vijay, Kristina Weitzel, Julia Cooper, Eric W. Atkinson, Jason E. Coleman, Kevin Otto, Martha Campbell Thompson 2020. Human Pancreas PACT Optical Clearing and High Resolution 3D Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.9gbh3sn Copy   

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