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Sample GSM3734913 Query DataSets for GSM3734913
Status Public on Sep 25, 2019
Title Splenic_aCD40_IL4_1
Sample type SRA
Source name Splenic mature B cells
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: AID-deficient splenic mature B cells
genotype: AID-deficient primary splenic B cells
treatment: with aCD40/IL4 stimulation
Treatment protocol Purified B cells were stimulated with either aCD40/IL4 for 48 hrs; CH12F3 cell lines were cultured in lymphocyte medium R15 and stimulated with anti-CD40+IL4+TGF-β for 24 hrs.
Growth protocol RPMI1640+10% FBS
Extracted molecule total RNA
Extraction protocol Briefly, 10 million cells were collected and permeabilized with the buffer (10 mM Tris-HCl pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, protease inhibitors and Rnase inhibitor). The permeabilized cells were resuspended in 100 ml of storage buffer (10 mM Tris-HCl pH 8.0, 25% (V/V) glycerol, 5 mM MgCl2, 0.1 mM EDTA and 5 mM DTT) for nuclear run-on with 2X run-on mix (5 mM Tris-HCl PH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.5 mM BrUTP, RNase inhibitor, 1% sarkosyl) at 37o for 5 min. RNA was extracted by Trizol and followed by hybrolyzation with NaOH at a final concentration of 0.2 N on ice for 18 min. After quenching with ice-cold Tris-HCl PH6.8 at a final concentration of 0.55 M and exchanging buffer via Bio-Rad P30 columns, the RNA was incubated with Br-dU antibody-conjugated beads (Santa Cruz biotechnology, sc-32323-ac) for 1 hr. The enriched run-on samples were incubated with RppH (NEB, M0356S) and hydroxyl repair with T4 PNK (NEB, M0201S), followed by ligating the 5’ and 3’ RNA adaptor. RT-PCR was performed from the adaptor-ligated RNA to obtain cDNA
Half of the cDNA was subjected to making Groseq libraries by two rounds of PCR with barcode primers. 200-500 bp products from the first round of PCR were subjected to the second round of PCR with the number of PCR cycles determined by test PCR amplification. The second round of PCR products were size-selected by SPRIselect beads (Beckman Coulter, B23318).
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model NextSeq 550
Description Run-on RNA
Data processing Library strategy: GRO-Seq
Reads were aligned using bowtie 2.2.8 with --non-deterministic flag
reads were quantified for specific loci using samtools version 1.8
wig files were produced using RseqQ package
Genome_build: mm9
Supplementary_files_format_and_content: wig files contain peak information for samples
Submission date Apr 24, 2019
Last update date Sep 26, 2019
Contact name Frederick W Alt
Organization name Boston Children's Hospital
Department PCMM
Lab Alt
Street address 1 Blackfan Circle
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
Platform ID GPL21626
Series (2)
GSE130266 Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching [GRO-Seq]
GSE130270 Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching
BioSample SAMN11491237
SRA SRX5730207

Supplementary file Size Download File type/resource 635.9 Mb (ftp)(http) BW 649.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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