Protocols   Select Another Resource Report Type

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol is used to dissociate adult (10 week) CD-1 mouse liver "on ice", preserving authentic gene expression profiles. The first layer consists of 3.5 hours rotation at 4 °C in 0.25% trypsin, and the second layer consists of 15 min. in bacillus licheniformis, with trituration on ice. The yield is 5000 cells/mg with 97% viability (as measured by trypan blue). 

Proper citation: Andrew Potter 2018. Adult mouse liver dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.r8id9ue Copy   

•   Source:

Authors: Daniel Gyllborg, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: Protocol for multiplexed in situ sequencing in tissue sections as an image-based spatial transcriptomic method. This is the second iteration of In Situ Sequencing (HybISS: Hybridization based In Situ Sequencing) based on the principles published in Ke et al. Nature Methods, 2013 and more recently Qian et al. Nature Methods, 2019. Here we present a modified method in detection chemistry using sequencing by hybridization approach for combinatorial decoding. This results in a more robust method for detecting larger probe panels for a more high-throughput in situ sequencing method.

Proper citation: Daniel Gyllborg, Mats Nilsson 2020. HybISS: Hybridization-based In Situ Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.xy4fpyw Copy   

•   Source:

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. 5: User-friendly protocol: SABER RNA FISH in cells. protocols.io https:// Copy   

•   Source:

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, and is an extension of the Frankenstein (D.O.F means Daughter Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® continuous gradient to remove debris. It is the alternative protocol when FACS is not available. This protocol works better with smaller samples. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions.

Proper citation: Luciano Martelotto 2021. Daughter of Frankenstein protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® continuous gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.bs99nh96 Copy   

•   Source:

Authors: Xingqi Chen, Ulrike Litzenburger, Yuning Wei, Alicia N. Schep, Edward L. LaGory, Hani Choudhry, Amato J. Giaccia, William J. Greenleaf, Howard Y. Chang
Group: Human Cell Atlas Method Development Community, HYChangLab
Summary: Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. PiATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1A protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

Proper citation: Xingqi Chen, Ulrike Litzenburger, Yuning Wei, Alicia N. Schep, Edward L. LaGory, Hani Choudhry, Amato J. Giaccia, William J. Greenleaf, Howard Y. Chang 2018. Pi-ATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.q6gdzbw Copy   

•   Source:

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, and is an extension of the Frankenstein (D.O.F means Daughter Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® continuous gradient to remove debris. It is the alternative protocol when FACS is not available. This protocol works better with smaller samples. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions.

Proper citation: Luciano Martelotto 2021. Daughter of Frankenstein protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® continuous gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.bstznep6 Copy   

•   Source:

Authors: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This protocol outlines the steps and methods for blood processing for PBMC to assist the MGH COVID-19 effort.

Proper citation: Moshe Sade-Feldman, Miguel Reyes, Alexandra-Chloe Villani 2020. MGH COVID-19 Effort Blood Processing Protocol for PBMC, Neutrophils and Plasma Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhnkj5e Copy   

•   Source:

Authors: Michael Hagemann-Jensen, Christoph Ziegenhain, Ping Chen, Daniel Ramsköld, Gert-Jan Hendriks, Anton J.M Larsson, Omid R. Faridani, Rickard Sandberg
Group: Human Cell Atlas Method Development Community

Proper citation: Michael Hagemann-Jensen, Christoph Ziegenhain, Ping Chen, Daniel Ramsköld, Gert-Jan Hendriks, Anton J.M Larsson, Omid R. Faridani, Rickard Sandberg 2020. Smart-seq3 Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bcq4ivyw Copy   

•   Source:

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 Quantification and Purity Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.si3ecgn Copy   

•   Source:

Authors: Robert Mahoney
Group: Human Cell Atlas Method Development Community
Summary: This protocol is used to dissociate adult (8-10 week) mouse testis into a single cell suspension. The entire procedure is carried out 'on ice', reducing artifact gene expression changes.  The yield is 5.4 million cells from 25 mg tissue and the viability is ~99%. 

Proper citation: Robert Mahoney 2018. Adult mouse testis cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.smkec4w Copy   

•   Source:

Authors: Benjamin Emert
Group: Human Cell Atlas Method Development Community, RajLab
Summary: Protocol for add azide modified nucleotide to 3' end of unmodified DNA olgionucleotide using terminal transferase. Protocol adapted from Winz et al. 2015. 

Proper citation: Benjamin Emert 2018. 3' Azide labelling with TdT. protocols.io https://dx.doi.org/10.17504/protocols.io.qnmdvc6 Copy   

•   Source:

Authors: Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard
Group: Human Cell Atlas Method Development Community
Summary: Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility and cost-efficiency can advance many research questions. Among the flexible plate-based methods, “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is one of the most sensitive and efficient ones. Here, we systematically evaluated experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be  the most sensitive and one of the most efficient and flexible scRNA-seq methods to date.

Proper citation: Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard 2018. mcSCRB-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.p9kdr4w Copy   

•   Source:

Authors: Elliott Swanson, Lucas Graybuck, Pete Skene
Group: Human Cell Atlas Method Development Community
Summary: TEA-seq is a method for Transcriptomic, Epitope, and Accessibility measurement from thousands of single cells on the 10x Genomics Multiome platform. It generates scRNA-seq, scATAC-seq, and cell surface epitope (BioLegend TotalSeq) sequencing libraries linked by 10x cell barcodes for coordinated analysis. TEA-seq has been optimized for use with peripheral blood mononuclear cells (PBMCs).

Proper citation: Elliott Swanson, Lucas Graybuck, Pete Skene 2020. TEA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bqagmsbw Copy   

•   Source:

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, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.

Proper citation: Luciano Martelotto 2021. S.O.F protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® discontinuous gradient . protocols.io https://dx.doi.org/10.17504/protocols.io.bsxhnfj6 Copy   

•   Source:

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

Proper citation: Adam Hunter 2018. CGAP Human Lung Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.qz9dx96 Copy   

•   Source:

Authors: Lira Mamanova
Group: Human Cell Atlas Method Development Community
Summary: This SOP describes the procedure for the in-house automated Smart-Seq2 workflow with off-the-shelf reagents. This set up utilises the benchtop robots and significantly reduces the experimental cost, hands-on time, as well as increase the reproducibility and workflow efficiency, while delivering a throughput of thousands of cells per day.Following library construction, samples are pooled in equivolume and quantified, prior to sequencing on the Illumina platform.

Proper citation: Lira Mamanova 2020. In-house automated Smart-Seq2. protocols.io https://dx.doi.org/10.17504/protocols.io.bnzsmf6e Copy   

•   Source:

Authors: Christian Pfleger, Shin Lin
Group: Human Cell Atlas Method Development Community
Summary: This protocol is for nuclear extraction from human heart tissue for single cell sequencing.

Proper citation: Christian Pfleger, Shin Lin 2020. Protocol for nuclear extraction from human heart tissue for single cell sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bcjqiumw Copy   

•   Source:

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

Proper citation: Adam Hunter 2018. CGAP Freezing Human Tissue in Isopentane. protocols.io https://dx.doi.org/10.17504/protocols.io.qz7dx9n Copy   

•   Source:

Authors: Satija Lab
Group: Human Cell Atlas Method Development Community, Satija Lab
Summary: This protocol presents a simple modification of the SMART-seq2 (Picelle et al, 2014) and SCRB-seq (Soumillon et al, 2014) protocols, with the goal of combining the sensitivity of SMART-Seq2 with the improved cost and throughput of 3' single cell protocols. Reverse transcription (RT) and PCR proceed as in SMART-Seq2, but using 3' barcoded oligos from SCRB-seq. While most barcoded methods (including SCRB-seq) pool cells together after RT, we perform PCR individually in each well, as this negates the need for an additional exonuclease and purification step, and therefore maintains the same molecular sensitivity as SMART-Seq2. However, we pool amplified cDNA from single wells immediately after PCR, and generate a single tagmentation-based 3' cDNA library. While this protocol therefore has advantages in both sensitivity and throughput, the absence of an exonuclease step prior to PCR disables the use of unique molecular identifiers for quantification.

Proper citation: Satija Lab 2018. Barcoded Plate-Based Single Cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.nkgdctw Copy   

•   Source:

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.bmcek2te Copy   

•   Source: