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Sample GSM3734846 Query DataSets for GSM3734846
Status Public on Sep 25, 2019
Title i3CBEc_sti_iEm_1-2
Sample type SRA
 
Source name CH12F3 murine lymphoma cells
Organism Mus musculus
Characteristics cell type: CH12F3 murine lymphoma cells
genotype: i3CBEc-AID-/-
activation: aCD40/IL4/TGFb
treatment: cytokine stimulation
target locus: AID-/-; non-coding allele deletion; three CBE insertion with the convergent orientation as 3'CBE downstream of Cg2a
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 genomic DNA
Extraction protocol About 10 million cells were crosslinked with 2% formaldehyde for 10 minutes at room temperature and quenched with glycine at a final concentration of 125 mM. Then, the crosslinked cells were lysed in the 3C lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, protease inhibitors) and nuclei were digested with NlaIII enzyme (NEB, R0125) at 37 ℃ overnight. They were then brought to 16℃, 100U of T4 ligase added (Promega, M1801) and they incubated again overnight at 16℃. The ligated products were de-crosslinked with Proteinase K (Roche, #03115852001) at 56 ℃ overnight and the 3C templates were purified by phenol/chloroform.
Libraries were prepared using 3C samples by HTGTS (Jane et al., 2018).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiSeq
 
Data processing Library strategy: 3C-HTGTS
Standard basecalling formats for Miseq reads
Miseq reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to the mm9 reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt and bait sequences shorter than 50nt.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph files were generated for displaying (Jane et al., 2018)
 
Submission date Apr 24, 2019
Last update date Sep 26, 2019
Contact name Frederick W Alt
E-mail(s) jianqiao.hu@childrens.harvard.edu
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 GPL16417
Series (2)
GSE130263 Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching [3C-HTGTS]
GSE130270 Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching
Relations
BioSample SAMN11491193
SRA SRX5730134

Supplementary file Size Download File type/resource
GSM3734846_i3CBEc_sti_iEm_1-2.bedgraph.gz 333.0 Kb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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