|
| Status |
Public on Jun 20, 2019 |
| Title |
BHLCVTBBXX-6 |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood IgG+ memory B cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood
cell type: CD19+ IgG+ memory B cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Smart-seq2 extraction protocol from Picelli et al, Nature Protocols 2014 (doi:10.1038/nprot.2014.006)
RNA was bead-purified from individual cells followed by cDNA synthesis by reverse transcription, template switching and preamplification using KAPA HiFi HotStart ReadyMix. Indexed libraries were then made by tagmentation and pooled. Typically, libraries from 75-80 cells were pooled per lane and sequenced with an Illumina HiSeq 3000/4000 paired-end cluster kit on a HiSeq 4000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
95 cells from donor VRC315.06, see README for details
|
| Data processing |
Samples were demultiplexed (see README file) using a custom Java application with a index read PHRED score threshold of 20
Adapters were removed using Trimmomatic-0.32 with parameters `ILLUMINACLIP:nextera.fa:2:30:10:4:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:50`
Reads were mapped against a database of human repeat regions using bowtie 2.2.5, and aligned reads were discarded.
Remaining reads were mapped against the hg38 reference using STAR 2.4.2a.
Read counts per gene were quantified using HTSeq 0.6.1 with parameters `-f bam -m union -r pos -i gene_name -a 10 --stranded no`.
PCA of read counts for all genes were used to identify outlier cells that were excluded from downstream analysis.
MiXCR 2.0 was used with suggested parameters for RNAseq data to reconstruct V(D)J junctions and cells with reported T cell receptor recombinations were removed from downstream analysis.
BraCeR (commit 28e101c) was used to reconstruct full-length BCR sequences and do clonal analysis.
Seurat 2.2.1 was used to analyze gene count data following the recommended practices in the tutorials.
Genome_build: hg38
Supplementary_files_format_and_content: CSV files containing one row per cell and one column per gene. Data is number of reads mapped to each gene in each cell.
|
| |
|
| Submission date |
Jun 18, 2019 |
| Last update date |
Jun 20, 2019 |
| Contact name |
Chaim A Schramm |
| Organization name |
NIAID, NIH
|
| Department |
Vaccine Research Center
|
| Lab |
Genome Analysis Core
|
| Street address |
40 Convent Dr
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE132923 |
Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly Generated Human Memory B-cell Responses to Influenza Hemagglutinin |
|
| Relations |
| BioSample |
SAMN12086512 |
| SRA |
SRX6088001 |
