An encapsulated lymphatic organ through which venous blood filters.
Attributes by original data submitter
Sample
source_name
Splenic
strain
C57BL/6
tissue
Spleen
antibody
None (Input DNA)
time
48h
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
B cells were cross-linked with 1 % formaldehyde at room temperature for 10 minutes and cross-linking was quenched by 125 mM glycine. Cells were lysed in SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) and the chromatin was sonicated to an average fragment length of 500 base pairs (bp) using a Branson Sonifier 450. Sonicated chromatin was pre-cleared and incubated with 2 µg of anti-RAD21 antibody (ab992, Abcam), 4 µl anti-CTCF (07729, Millipore), 2 µg anti-PAX5 (sc-1974, Santa Cruz) and 1 µg anti-IgG (sc-2027, Santa Cruz) overnight. The complexes were washed with increasing salt stringency and cross-links were reversed in the presence of 0.2 M NaCl at 65 °C for 6 hours. Proteinase K treatment was carried out in the presence of 10 µM EDTA, 40 µM Tris-HCl, pH 6.5 and 20 µg Proteinase K. DNA fragments were recovered with phenol-chloroform extraction following one chloroform extraction. DNA was precipitated with 2 volumes of ethanol, 1/3 volumes of 7.5 M ammonium acetate and 20 µg glycogen with an overnight incubation at -20 °C. Precipitated DNA was washed with 70 % Ethanol to remove excess salt and DNA was resuspended in 30 µl TE buffer supplemented with 0.3 µg RNAseA. Immunoprecipitated fragments were end-repaired with NEBnext End Repair Module (NEB) and purified by using Agencourt Ampure XP - beads (Beckman Coulter) and A-tailed using the NEBnext dA-Tailing Module according to manufacturers’ instructions. After purification, adaptors were ligated (Adaptor-Oligo 1: 5'ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3', Adaptor-Oligo 2: 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3') by using 1x NEBnext Quick Ligation Buffer (NEB), 10x excess of DNA Adaptors, 5 µl Quick T4 DNA Ligase (NEB) at 50 µl total volume. Large-scale amplification of library constructs was performed by using the PCR Enrich Adaptor Ligated cDNA Library module (NEB).