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GEO help: Mouse over screen elements for information. |
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Status |
Public on Mar 20, 2012 |
Title |
RNA-seq mature B [10426] |
Sample type |
SRA |
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Source name |
B-1a B
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Organism |
Mus musculus |
Characteristics |
genotype/variation: Cd79a-Cre R26CA(Ebf1/+) strain: C57BL/6 chip antibody: NA extraction method: Ex vivo (FACS sort) tissue: Spleen
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Extracted molecule |
total RNA |
Extraction protocol |
RNA-Seq: RNA was isolated from ex vivo sorted cells using the RNeasy Plus Mini Kit (Qiagen), and mRNA was obtained by two rounds of poly(A) selection using the Dynabeads mRNApurification kit (Invitrogen) followed by fragmentation by heating at 94oC for 3 min. The fragmented mRNA was used as template for first-strand cDNA synthesis with random hexamers using the SuperScript VILOTM cDNA Synthesis Kit (Invitrogen). dNTPs were removed on a Mini Quick Spin Column (Roche) prior the second-strand cDNA synthesis with 100 mM dATP, dCTP, dGTP and dUTP in the presence of RNase H, E. coli DNA polymerase I and DNA ligase (Invitrogen). The incorporation of dUTP allowed elimination of the second strand during library preparation. ChIP-seq: Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina Genome Analyzer |
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Data processing |
Illumina GA Pipeline RTA 1.8.7; ChIP-seq : Sequence reads that passed the Illumina quality filtering were considered for alignment against the mouse genome assembly version of July 2007 (NCBI37/mm9) using the Bowtie program version 12.5 (Langmead et al, 2009), allowing up to two mismatches and ignoring any read that would map more than once in the genome. In detail, the alignment was done using the following parameters: –m 1 –v 2 –best –strata –tryhard. RNA-Seq cleaning was performed using the following settings: –v 3 –k 1 --tryhard --chunkmbs 256 The RNA-seq samples were cut down to a common read length (30bp) and aligned to the mouse transcriptome (mm9) using TopHat version 1.3.1.(1). The number of reads per gene was counted using HTseq version 0.5.3 with the overlap resolution mode option set to "union". Genome Build: 10426.30.counts.tab: mm9
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Submission date |
Feb 16, 2012 |
Last update date |
May 15, 2019 |
Contact name |
Meinrad Busslinger |
E-mail(s) |
busslinger@imp.ac.at
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Phone |
00431-79730
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Organization name |
Instutute of Molecular Pathology
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Street address |
Dr. Bohrgasse 7
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City |
Vienna |
ZIP/Postal code |
1030 |
Country |
Austria |
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Platform ID |
GPL9185 |
Series (1) |
GSE35857 |
Essential role of EBF1 in B cell immunity by controlling the generation and function of distinct mature B cell types |
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Relations |
SRA |
SRX120216 |
BioSample |
SAMN00790692 |
Supplementary file |
Size |
Download |
File type/resource |
GSM876634_10426.30.counts.tab.gz |
178.2 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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