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Sample GSM4028041

Query DataSets for GSM4028041

Status

Public on Aug 13, 2019

Title

BCR6__S0170_S170

Sample type

SRA

Source name

IGHV1-69_VL_Post Boost 3_Splenic B cells

Organism

Mus musculus

Characteristics

genotype: IGHV1-69
antibody chain: VL
experiment: Post Boost 3
cell type: Splenic B cells
molecule subtype: BCR Amplicon from RNA

Extracted molecule

total RNA

Extraction protocol

Mouse spleens were removed and single cell suspensions were obtained using 70 um cell strainer. Cells were stained using Aqua Live/Dead dye, followed by a cocktail of the following antibodies with the relevant probes: CD4 Alexa Fluor 700 (Cat# 557956, BD Biosciences); and CD3e PE-Cy7 (100320, Biolegend), CD19 BV421 (Cat# 115549, BioLegend), IgM BV605 (Cat# 406523, BioLegend), IgG PerCPCy5.5 (Cat#405314, Biolegend). Naïve B cells (CD3-/CD19+/IgG-/IgM+) were bulk sorted into RLT/1% beta-mercaptoethanol (BME). SS-np induced single B cells, including post boost 1, post boost 2 and post boost 3 (CD3-/CD19+/IgG+/IgM-/H1+ probe/H5+ probe), were sorted into 96 well plates containing RLT lysis buffer/1% BME. SS-np on-target single B cells were defined as CD3-/CD19+/IgM+/IgG-/SS-np Alexa 488+ probe/SS-np Alexa 594+ probe/SS-npDstem Alexa 647- probe/SS-npDstem Alexa 546- probe. Adoptive transfer single B cells are defined as CD3-/CD4-/B220+/GL7+/CD45.1-/CD45.2+/G6+.
RNA seq BCR libraries from single cells were generated by whole transcriptome amplification (WTA) using the Smart-Seq2 protocol (Trombetta et al., 2014). Single cell BCR amplicons for heavy and light chains (FR1 to CDR3) were generated separately using a pool of partially degenerate V region gene primers against all possible IGHV (human) or IGLV (mouse) and IGKV (mouse) segments in the FR1 region and reverse primers against the heavy or light constant regions containing the Illumina P7 and P5 sequences. Barcodes and Illumina sequencing adaptors (based on Nextera XT Index Adapters, Illumina Inc.) were then added to each amplified heavy and light chain using a step-out PCR. Single-cell BCR libraries were sequenced using paired end 250x250 reads and 8x8 index reads on an Illumina MiSeq System (MiSeq Reagent Kit v2 500-cycle; Illumina, San Diego, US). Bulk BCR amplicons were generated following the same WTA step and two subsequent PCR steps as above, except that in the first PCR, the cocktail of V gene primers was replaced with a forward primer specific for the FR3 region within the VH gene of interest (IGHV1-69 or IGHV1-2). The resulting product (FR3 to CDRH3) was sequenced on Illumina MiSeq as above except without index reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

processed data file: Consensus_Alignments.txt

Data processing

For single cells, paired end reads were merged into full length heavy and light chain reads using PANDAseq (v2.7).
Full length reads were aligned/constructed from human heavy and light chains using MigMAP (v.1.0.2).
Consensus heavy and light chains for each single cell were then determined by collapsing all reads by CDR3 sequence and calling the top hit for heavy and light, respectively. Any sequence without at least 25 reads or frequency 2x greater than the next sequence of the same chain was denoted without consensus.
For bulk heavy chain analysis, reads were aligned to human using mixcr (v.2.1.8) with the following parameter for alignment: OvParameters.geneFeatureToAlign = {FR3Begin:Vend}.
Downstream bulk analysis was preformed in R (v.3.4.3) using the tcR package (v2.2.3) and custom scripts.
Genome_build: IMGT (compiled Dec. 2017)
Supplementary_files_format_and_content: tab delimited txt file

Submission date

Aug 12, 2019

Last update date

Aug 14, 2019

Contact name

Maya Sangesland

Organization name

Ragon Institute of MGH, MIT, and Harvard

Lab

Lingwood Lab

Street address

400 Technology Square