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Sample GSM4225089 Query DataSets for GSM4225089
Status Public on Jan 11, 2020
Title ESC_line2_Exp2
Sample type SRA
Source name Blastocyst derived embryonic stem cells line 2
Organism Mus musculus
Characteristics strain: C57BL/6/129SVE
cell type: non-homologous end-joining deficient
genotype: Xrcc4-/- p53-/-
Treatment protocol none
Growth protocol embryonic stem cells: DMEM medium (Corning, 10-013-CV) supplemented with 15% FBS
embryonic stem cells-derived neural progenitor cells : N2B27+ 1% B27 (without retinyl acetate) for differentiation and NBBG + 2% B27 (without retinyl acetate) for culture maintainance
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) or gel PAGE purification
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 3000
Description without APH treatment
Data processing Library strategy: GRO-Seq
Reads were aligned using bowtie 2.2.8 with --non-deterministic flag
RPKM values were calculated with GFOLD V1.1.2
wig files were produced using MACS2
Genome_build: mm9
Supplementary_files_format_and_content: wig files contain peak information for samples
Submission date Dec 18, 2019
Last update date Jan 11, 2020
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 GPL21493
Series (2)
GSE142312 Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture [GRO-seq]
GSE142315 Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture
BioSample SAMN13630734
SRA SRX7413438

Supplementary file Size Download File type/resource 96.6 Mb (ftp)(http) BW 100.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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