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Sample GSM2891869

Query DataSets for GSM2891869

Status

Public on Nov 19, 2018

Title

KO_8E_704508

Sample type

SRA

Source name

B cells

Organism

Mus musculus

Characteristics

tissue: bone marrow cell
cell type: B cells
tag: P198A/P198A mutant

Treatment protocol

BM cells from straight chimeras were stained with B220,CD43,CD45.1,CD45.2,CD24,BP-1,IgM and IgD Abs.Cells were sorted using a FacsARIA III (BD), using the following gating strategy: viable, B220+CD43+CD45.1-CD45.2+CD24+BP1- (FrB/pro-B); B220+CD43-CD45.1-CD45.2+IgM-IgD- (FrD/late preB) and viable, B220+CD43-CD45.1-CD45.2+IgM+IgD- (FrE/Immature)

Growth protocol

BM cells of WT and P198A/P198A mutant mice (generated by CRISPR/Cas9) were injected into lethally irradiated CD45.1 mice to generate straight chimeras. After 8 weeks reconstitution, 5 WT mice and 5 P198A/P198A mutant straight chimeras mice BM were freshly isolated for the experiment.

Extracted molecule

polyA RNA

Extraction protocol

100 cells per sample were directly sorted into ice cold cell lysis buffer (0.4% (vol/vol) Triton X-100 and 2 U/μl RNase inhibitor, 4 x107 dilution of ERCC spike in control, comprising a pre-formulated blends of 92 transcripts, derived and traceable from NIST-certified DNA plasmids), 2.5mM dNTPs (Thermo-Fisher), 2.5µM Oligo (Oligo-dT30VN.) and immediately frozen in dry ice.Sorted cells were processed using the Smart-seq2 protocol with minor changes
Indexing of the samples was performed using the Nextera XT DNA Sample Preparation Index kit (24 index primers, manufacturer (Illumina)), according to manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Bioinformatic analysis was performed using CGAT Pipelines and tools (https://github.com/CGATOxford/CGATPipelines). Reads were aligned to the mm10 mouse genome using hisat2 version 2.1.0, and reads were quantified over feature annotations (ensemble81) using featureCounts program version 1.4.6 within the Subread software package. DESeq2 was used for statistical analysis of the differential expression for each gene between wild-type and ZIP7P198A/P198A mutants.
Genome_build: mm10

Submission date

Dec 18, 2017

Last update date

Nov 19, 2018

Contact name

Adam Cribbs

E-mail(s)

[email protected]

Organization name

University of Oxford

Department

NDORMS