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Sample GSM3309449 Query DataSets for GSM3309449
Status Public on Oct 01, 2018
Title WT pre-B cells RNA #1
Sample type SRA
 
Source name C57BL/6 pre-B cells
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: Bone marrow
genotype: WT C57BL/6
Extracted molecule total RNA
Extraction protocol Mouse bone marrow cells were enriched for CD19+ B cells using Miltenyi CD19 microbeads (mouse). CD19+ B cells were sorted to either small pre-B cells (CD2+, CD43-) or pro-B cells (CD2-, CD43+).
For gDNA, sonicated fragments were end repaired, A-tailed and adapter ligated. Jš›‹ gene biotinyated primer extension products were pulled out with streptaviding. Sequential PCRs added index, barcode and flow cell binding sequences. The last PCR utilized NEBNext Dual indexing primers. For RNA, cDNA was made using Transcriptor high-fidelity reverse transcriptase (Roche). RNA was degraded and ligated to bridge adapters. Sequential PCRs were employed as in gDNA.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina MiSeq
 
Description WT pre-B cells RNA biological replicate #1
Data processing Library strategy: VDJ-seq
Demultiplexed paired-end reads were adapter trimmed and quality filtered (Phred>20) using TrimGalore (max of 50 bases trimmed from 3ā€™ end of read, if more bases trimmed from either read then both reads of the pair were thrown out).
Paired-end reads were then merged using pandaseq using default settings. VJ gene calling was performed on merged reads using Abstar (https://github.com/briney/abstar) with a custom version of the minimal output setting, appending Vk gene length in the output.
In addition, a Vk gene reference file was custom made for C57BL/6 (*01 alleles) and each Vk gene was cross-referenced to the mouse genome build mm9 on UCSC genome browser.
Abstar output was processed with a custom R pipeline (https://github.com/salvatoreloguercio/RepSeqPipe) which computed Vk or Jk gene usage statistics. The cutoff for Vk gene assignment was a minimum read length of 150 bp and a minimum of 95% sequence identity. Reads passing this filter were then deduplicated based on the six random adapter nucleotides (gDNA and RNA) and in the case of gDNA, samples were additionally deduplicated based on the starting position of the Vk gene read which are random due to shearing. Reads that contained the same start site (for gDNA) and random 6Ns were presumed to have originated from the same fragment and only counted once. For gDNA, reads needed further processing to deal with Jk PCR primer cross-amplification in order to accurately re-assign Jk gene calling. gDNA reads were re-assigned to their proper Jk gene based on the sequence upstream of each Jk primer. This did not include the most Vk proximal nucleotide of the Jk exon to allow for potential VkJk junctional loss. One pre-B cell gDNA library was prepped using a second set of Jk PCR primers located further downstream of the original Jk primers (closer to the biotin). Since these Jk primers were further away from the VkJk junction, we excluded the 6 most Vk proximal nucleotides from the Jk exon in Jk gene identification.
Genome_build: mm9
Supplementary_files_format_and_content: Microsoft Excel spreadsheet with normalized read counts / percent read counts for 140 Vk genes.
Supplementary_files_format_and_content: Columns: 1) Vk gene name; 2) total.occurrences: # total reads; 3) X..total.occurrences: % total reads; 4) v_genes_prod: # productive reads; 5) prod_total: % productive reads within total reads; 6) prod_prod: % productive reads within all productive reads; 7) v_genes_nonprod: # non-productive reads; 8) nonprod_total: % non-productive reads within total reads; 9) nonprod_nonprod: % non-productive reads within all non-productive reads
 
Submission date Jul 27, 2018
Last update date Oct 01, 2018
Contact name Salvatore Loguercio
Organization name TSRI
Department MB
Lab Balch
Street address 10550 North Torrey Pines Rd
City La Jolla
State/province California
ZIP/Postal code 92121
Country USA
 
Platform ID GPL16417
Series (2)
GSE117800 Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vk Gene Rearrangement in Pre-B Cells and Pro-B Cells [VDJ-seq]
GSE117804 Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vkappa Gene Rearrangement in Pre-B Cells and Pro-B Cells
Relations
BioSample SAMN09728213
SRA SRX4477567

Supplementary file Size Download File type/resource
GSM3309449_WT_pre-B_cells_RNA_1.xlsx 54.9 Kb (ftp)(http) XLSX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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