|
| Status |
Public on Feb 21, 2020 |
| Title |
SPF-3_PP_IgH_GC |
| Sample type |
SRA |
| |
|
| Source name |
Peyer's Patches germinal center B cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
condition: SPF
tissue: Peyer's Patches
locus (ig heavy-chain or light-chain): IgH
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Spleen and PPs were dissected out from 8-12 week old mice, prepared into single cell suspensions and purified by EasySep® Negative Selection B cell Enrichment Kit (Stem Cell Techonologies) according to the manufacturer’s protocol. Purified B cells were stained with anti-B220-PE (1:2000) (eBiosciences), anti-CD38-APC (1:200) (eBiosciences) and anti-GL7-FITC (1:200) (Biolegend). GC (B220+GL7+CD38-) and nonGC (B220+GL7-CD38+) B cells were sorted from the same sample by fluorescence-activated cell sorting (FACS). Genomic DNA from sorted cells was prepared using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol.
LAM-HTGTS-Rep-SHM-Seq was derived from HTGTS-Rep-seq (Lin et. al., PNAS 2016) with several modification. All libraries were made using 400 ng genomic DNA as starting materials, so that the level of PCR amplification was kept the same among different samples. In the cases when less than 400 ng genomic DNA was obtained from GC or non-GC B cell samples, genomic DNA from JM8a3, a C57BL/6 ES cell line, was added to make the starting DNA template amount consistent among samples. The library preparation procedure was mostly done as previously described with a few modifications in the linear amplification-mediated PCR step, where we used one reaction of 50 μl instead of 8 reactions and performed 100 cycles instead of 80 cycles. scRNA-seq libraries were prepared using 10x Genomics Library Kits and sequenced on an Illumina NextSeq500 according the manufacturer’s recommendations.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Library strategy: LAM-HTGTS-Rep-SHM-Seq
LAM-HTGTS-Rep-SHM-Seq data analysis was done by the LAM-HTGTS-Rep-SHM-Seq bioinformatics pipeline (Github address: https://github.com/Yyx2626/HTGTSrep), using IgBLAST with default parameters for qualified reads mapping. Only reads with more than 98% of the sequences having a sequencing Quality Score ≥20 were kept; only joined reads (R1 and R2 had an overlap region ≥10 bp and mismatch rate ≤8%) were kept.
scRNA-seq raw sequencing files were aligned to the mouse V(D)J sequence using Cell Ranger: V(D)J Pipelines (10x Genomics) and the V usage and mutation profiles were generated and visualized by Loupe V(D)J Browser.
Genome_build: mm9
Supplementary_files_format_and_content: .db.xls files were generated using LAM-HTGTS-Rep-SHM-Seq pipeline (IgBLAST); .vloup files were generated using Cell Ranger: V(D)J Pipelines (10x Genomics).
|
| |
|
| Submission date |
Nov 21, 2019 |
| Last update date |
Feb 21, 2020 |
| Contact name |
Frederick W Alt |
| Organization name |
Boston Children's Hospital / Harvard Medical School
|
| Department |
Program in Cellular and Molecular Medicine (PCMM)
|
| Lab |
Frederick Alt
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE140795 |
Peyer’s Patch Germinal Centers Select Innate-like BCRs |
|
| Relations |
| BioSample |
SAMN13348279 |
| SRA |
SRX7199805 |
