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Sample GSM4176370 Query DataSets for GSM4176370
Status Public on Jan 01, 2020
Title ChIP_seq_RAG2_PB_DJK
Sample type SRA
Source name Abelson-transformed pre-B cell
Organism Mus musculus
Characteristics cell type: Abelson-transformed pre-B cell
genotype: DJK
strain: C57BL/6
chip antibody: Anti-RAG2
chip antibody: rabbit polyclonal
Treatment protocol Pre-B cells were arrested in G1 by treatment with 3 mM imatinib for 48 hr.
Growth protocol Abelson pre-B cell lines 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).
Extracted molecule genomic DNA
Extraction protocol Twenty million preB cells were used for ChIP-seq.
Rabbit monoclonal antibodies for RAG1 (mAb 23) and RAG2 (mAb 39) (Coster et al., 2012) and a polyclonal rabbit antibody for H3K4me3 (Millipore) were used. Cells were fixed by adding 37% formaldehyde (F1635, Sigma) to a final concentration of 1% and incubated at 37°C for 10 min. Fixation was quenched by addition of 1M glycine (Sigma) in PBS at a final concentration of 125 mM. Twenty million fixed cells were washed twice with cold PBS and pellets were snap frozen in dry ice and stored at -80°C. Fixed cell pellets of 20 million cells were thawed on ice and resuspended in 2 mL of cold RIPA buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 Complete Mini EDTA free proteinase inhibitor (Roche)). Sonication was performed using the Covaris S220 sonicator at duty cycle 20%, peak incident power 175, cycle/burst 200 for 30 min at 4°C or using the Branson sonifier at amplitude 35%, 12 cycles of 20 s sonication and 30 s of pause at 4°C. Chromatin were clarified by centrifugation at 21,000 g at 4°C for 10 min and precleared with 80 ul prewashed Dynabeads protein A (ThermoFisher) for 30 min at 4°C. 40 μl prewashed Dynabeads protein A were incubated with 10 μg of each respective antibody in 100 μl of PBS for 10 min at room temperature in continuous mixing, washed twice in PBS for 5 min and added to 1 mL of chromatin followed by overnight incubation at 4°C on a rotator. Beads were then collected in a magnetic separator (DynaMag-2 Invitrogen), washed twice with cold RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (10 mM Tris pH 8.0, 1mM EDTA) plus 0.2% Triton X-100, and once with TE. Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1mg/mL of Proteinase K (Ambion). DNA was purified using Zymo ChIP DNA clean and concentrator kit (Zymo Research) and eluted in 20 μl. The entire ChIP DNA was used to prepare Illumina sequencing libraries. End-repair was performed in 75 μl of T4 ligase reaction buffer, 0.4 mM of dNTPs, 4 U of T4 DNA polymerase (NEB), 13.5 U of T4 Polynucleotide Kinase (NEB) and 1.5 U of Klenow fragment (NEB) at 24°C for 30 min in a ThermoMixer C at 400 rpm. End-repair reaction was cleaned using 2X Agencourt AMPure XP beads and eluted in 15 μl of EB that was used for A-tailing reaction in 30 μl of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 U of Klenow fragment exo- (NEB) at 37°C for 30 min. The 30 μl of the A-tailing reaction was mixed with Quick Ligase buffer 2X (NEB), 3,000 U of Quick ligase and 5 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 75 μl and incubated at 25°C for 20 min. The adaptor was prepared by annealing the following HPLC oligos: 5’-phosphate/GATCGGAAGAGCACACGTCT-3’ and 5’-ACACTCTTTCCCTACACGACGCT CTTCCGATC∗T-3’ (∗phosphorothioate bond). Ligation was stopped by adding 50mM of EDTA and cleaned with 1.8X Agencourt AM- Pure XP beads and eluted in 15ul of EB that was used for PCR amplification in a 50 uL reaction with 1 μM primers TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC∗T, TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T, (NNNNNNNN represents barcode and ∗ a phosphothiorate bond), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98°C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C . PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-500bp was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina Nextseq500 (75bp single-end reads).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model NextSeq 550
Data processing Reads were aligned to the GRCm38p2/mm10 genome assembly.
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 2 -m1 -l 50
Genome_build: GRCm38p2/mm10
Supplementary_files_format_and_content: bigwig files were made using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig
Submission date Nov 19, 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)
GSE140655 Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire [ChIP-seq]
GSE140677 Intra-Vk Cluster Recombination Shapes the Ig Kappa Locus Repertoire
BioSample SAMN13326880
SRA SRX7182808

Supplementary file Size Download File type/resource 191.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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