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Sample GSM4176074 Query DataSets for GSM4176074
Status Public on Jan 01, 2020
Title END_seq_THY
Sample type SRA
 
Source name Primary thymocyte
Organism Mus musculus
Characteristics cell type: Primary thymocyte
genotype: WT
strain: C57BL/6
Treatment protocol Pre-cells were arrested in G1 by treatment with 3 mM imatinib for 48 hr.
Growth protocol Abelson pre-B cell lines and primary mouse bone marrow pre-B cells were cultured DMEM supplemented 10% FBS, with 2 mM L-glutamine, 1 mM Sodium piruvate, non-essential amino acids, 0.05 mM β-mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 U/ml) (Invitrogen). Primary mouse bone marrow pre-B cells were cultured in the presence of recombinant IL-7 (ThermoFisher).
Extracted molecule genomic DNA
Extraction protocol Single cell suspensions of pre-B cells (30 million per agarose plug), primary mouse bone marrow pre-B cells (10 million per agarose plug), and thymocytes (70 million per agarose plugs) were washed in PBS, embedded in agarose and transferred into plug molds (Bio-Rad CHEF Mammalian Genomic DNA plug kit) and processed as previously described (Canela et al., 2016). Primary mouse bone marrow pre-B cells for END-Seq analyses were obtained from 6-12 week old Vavp-Bcl2-transgenic mice (Ortega-Molina et al., 2015) and cultured in the presence of recombinant IL-7 (ThermoFisher) for 7-10 days at 2 x 106 cells/ml in vitro. To induce cell cycle arrest, cells were resuspended in media without IL-7 and maintained at 2 × 106 cells/ml for 48 hours and harvested for END-seq.
END-seq was performed as described in Canela et al., 2017. Agarose plugs were made using the Bio-Rad CHEF Mammalian Genomic DNA plug kit. Embedded cells were lysed and digested using Proteinase K (50º C, 1 hour then 37ºC for 7 hours). Plugs were rinsed in TE buffer and treated with RNase A at 37ºC, 1 hour. The next enzymatic reactions were performed with the DNA in agarose plugs to prevent shearing. DNA ends were blunted for 1 hour at 25ºC with Exonuclease T (NEB). After blunting, A-tailing was performed, followed by ligation of “END-seq hairpin adaptor 1”, 5′-Phos-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[Biotin-dT]U[Biotin-dT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Agarose plugs were then melted and dialyzed and DNA was sonicated to a median shear length of 170bp using Covaris S220 sonicator. DNA was ethanol-precipitated and dissolved in 70 ul TE buffer. Biotinylated DNA was isolated using MyOne Streptavidin C1 Beads (ThermoFisher #650-01), followed by end repair and dA-tailing. The second end was ligated to “END-seq hairpin adaptor 2”, 5′-Phos-GATCGGAAGAGCACACGTCUUUUUUUUAGACGTGTGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Hairpins were digested using USER (NEB), and the resulting DNA fragments were PCR amplified using TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond). PCR fragments were isolated by size selection from agarose gel, selecting 200-500 bp fragments followed by DNA purification using QIAquick Gel Extraction Kit. Libraries were quantified using KAPA Library Quantification Kit and sequenced using Illumina NextSeq 500 or 550.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model NextSeq 550
 
Data processing Library strategy: END-seq
mouse and human END-seq reads were aligned to the GRCm38p2/mm10 and GRCh37/hg19 genome assemblies,respectively.
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 3 -k1 -l 50
END-seq peaks were called using MACS 1.4.3 with the parameters: --nolambda,-- nomodel and –keep-dup=all (keep all redundant reads). For Etoposide-treated END-seq data peak calling, we used the corresponding non-treated samples as control, keeping >10 fold-enriched peaks.
END-seq data from pre-B cells, where cells with ZFN break were spiked-in to the library at a 1:50 (2%) ratio, the RPKM value for each peak was divided by the signal around the spiked-in breaks and then multiplied by 2, to get values as cell-percentage. From this value, X, the #cells per lesion were calculated as 100/X.
Genome_build: GRCm38p2/mm10 for mouse and GRCh37/hg19 for human
Supplementary_files_format_and_content: bigwig files were made using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig
 
Submission date Nov 18, 2019
Last update date Jan 02, 2020
Contact name Yaakov Maman
Organization name Bar-Ilan University
Department Azrieli Faculty of Medicine
Lab Genome Instability & Cancer
Street address Henrietta Szold 8
City Safed
ZIP/Postal code 1311502
Country Israel
 
Platform ID GPL21626
Series (2)
GSE140635 Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [END-seq]
GSE140677 Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire
Relations
BioSample SAMN13326822
SRA SRX7182777

Supplementary file Size Download File type/resource
GSM4176074_END_seq_THY.bw 88.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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