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Sample GSM6969925

Query DataSets for GSM6969925

Status

Public on May 25, 2023

Title

Pooled BCR Library 2 for Donors HDL153, HDL141, HDL142

Sample type

SRA

Source name

mixture of lung lymph node, mesenteric lymph node, lung

Organism

Homo sapiens

Characteristics

tissue: mixture of lung lymph node, mesenteric lymph node, lung
cell type: B cells
antibodies/tags: none

Extracted molecule

polyA RNA

Extraction protocol

Lung and lymph nodes were mechanically and enzymatically digested into single-cell suspensions as described in Szabo et al, Nature Communications, 2019. B cells were enriched by FACS using antibodies against CD45, CD33, CD66b, CD3, and CD19 to isolate CD45+/CD66b-/CD33-/CD3-/CD19+ cells from each tissue. Enriched B cell populations from each sample were resuspended in PBS/FBS/EDTA and stained with 3 uL of a unique TotalSeq C DNA-barcoded hashtag for 30 minutes on ice. Cells were then washed, counted, and pooled in PBS/FBS to ensure equal nunbers of live cells from each sample.
CITE-seq/scBCR-seq libraries were constructed using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (10x Genomics) with both a 5’ Feature Barcode Kit (10x Genomics) for antibody-derived tag (ADT) amplification and a Chromium Single Cell Human BCR Amplification Kit (10x Genomics) for targeted BCR amplification, according to the manufacturer’s instructions.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Description

JoshGC.bcr.filtered_contig_annotations.csv.gz

Data processing

Quantification of scRNA-seq library pools was performed by pseudoalignment to the human transcriptome (Gencode v24 annotation derived from GRCh38) using kallisto v0.46.2 in “BUS” mode. ADT barcodes were extracted from demultiplexed fastq files using custom scripts (https://github.com/simslab/DropSeqPipeline8). Hashtag barcodes from cell-identifying barcodes containing >1,000 unique transcripts were demultiplexed using the Hashsolo with default parameters. scBCR-seq data were demultiplexed and aligned with VDJ cassette and variable sequencing identification using Cell Ranger VDJ v7.0.1 using the GRCh38/Ensembl v7.0.0 annotation from 10x Genomics.
Assembly: GRCh38/Gencode v24
Supplementary files format and content: CITE-seq/scRNA-seq files are tab-delimited count matrices where each column is a cell and each row is a feature (antibody or gene). There is a header indicating the cell-identifying barcode, hashtag, and sample-of-origin for each cell (column). The header is formatted as barcode_donor_hashtag_tissue.
Supplementary files format and content: scBCR-seq files are csv filtered contig files output from Cell Ranger VDJ that contain the VDJ cassettes, nucleotide/amino acid sequences of variable regions, cell-identifying barcode, hashtag, and sample-of-origin for each cell.
Library strategy: BCR-seq

Submission date

Jan 24, 2023

Last update date

May 25, 2023

Contact name

Peter A Sims

E-mail(s)

pas2182@columbia.edu

Organization name

Columbia University

Street address

3960 Broadway, Lasker 203AC