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| Status |
Public on Sep 25, 2019 |
| Title |
CH12F3NCdel_nonsti_1 |
| Sample type |
SRA |
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| Source name |
CH12F3 murine lymphoma cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: AID-deficient CH12F3 murine lymphoma cells
genotype: CH12NC-delta-AID-/-
treatment: without stimulation
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| 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.
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| Growth protocol |
RPMI1640+10% FBS
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| 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).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
Run-on RNA
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| 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
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| Submission date |
Apr 24, 2019 |
| Last update date |
Sep 26, 2019 |
| Contact name |
Frederick W Alt |
| E-mail(s) |
jianqiao.hu@childrens.harvard.edu
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| Organization name |
Boston Children's Hospital
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| Department |
PCMM
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| Lab |
Alt
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| Street address |
1 Blackfan Circle
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| 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 |
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| Relations |
| BioSample |
SAMN11491231 |
| SRA |
SRX5730213 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3734919_CH12F3NCDel_nonsti_1.neg.bw |
576.1 Mb |
(ftp)(http) |
BW |
| GSM3734919_CH12F3NCDel_nonsti_1.pos.bw |
588.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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