2025-12-03
ENA first public
ERC000011
ENA-CHECKLIST
Immuno-genomics & Systems Biology laboratory, Institute of Life Sciences (ILS), Bhubaneswar, Odisha, India-751023.
INSDC center name
public
INSDC status
empty shRNA
RNA interference
ERS28163670
SRA accession
E-MTAB-10992:Sample 21
Submitter Id
ArrayExpress
broker name
MutuDC1940
cell line
CD8_alpha-positive CD11b-negative dendritic cell
cell type
not collected
collection date
house mouse
common name
Protocols: The CD8α+ lines (mutuDC) were cultured and stable Empty (Control) and NCoR1 shRNA vector (NCoR1 KD) transduced cells were generated as described in previous studies (Fuertes Marraco et al., 2012; Pigni et al., 2018). Two  independent biological replicate of Control and NCoR1 KD cells were treated with CpG-B, poly(I:C) (pIC) and combined CpG+pIC for 2hr and 6hr. Cells were further taken for H3K27ac ChIP-seq. The ChIP experiments were performed as per the Mayer’s Lab Protocol. The cells were cross-linked using 1% formaldehyde (Sigma) for 10 min at room temperature followed by quenching the reaction using 2.5 M glycine (Sigma) for 10 min. The cells were lysed in the FARHAM lysis buffer and centrifuged at 2000rpm at 4°C for 8 min. The chromatin was fragmented using a Bioruptor (Diagenode) sonicator for 30 min using high amplitude and 30s ON & 30s OFF cycles to obtain 200-500 bp size fragments. The concentration of the chromatin was estimated using a NanoDrop (Thermo) and the chromatin was diluted with a RIPA buffer prepared without protease inhibitor to make 125μg/ml of chromatin for each IP. 30ul of Dyna Magnetic beads (Anti-rabbit) were taken and added to 1ml tube for each IP. 3ul of rabbit monoclonal anti-H3K27ac antibody (Abcam, cat no: ab-177178), were added and incubated at 4°C overnight on a rocker shaker. Next day, the beads were washed six to seven times with LiCl buffer (1% NP-40, 100mM Tris HCl (pH 7.5), 500mM LiCl, 1% Sodium Deoxycholate) followed by two washes with TE buffer (10nM Tris HCl (pH 7.5), 0.1mM EDTA (pH 8)). After removing the wash buffer completely, protein-bound chromatin complexes were eluted from beads for 30 min using 200ul of elution buffers. The eluted chromatin was reverse-crosslinked by overnight incubation on the shaker using 8ul of 5M NaCl. Next day DNA was purified from the reverse cross-linked chromatin by proteinase-K and RNase digestion followed by purification using PCR purification kit (Qiagen). H3K27ac ChIP sample library preparation was performed using an NEB ChIP library preparation Kit
description
adult
developmental stage
CD11c:SV40LgT
genotype
not collected
geographic location (country and/or sea)
CD8 positive; CD11chigh; B220 negative; DEC205 positive; CD24high, CD11b negative
immunophenotype
anti-H3K27ac
immunoprecipitate
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
spleen
organism part
2
replicate
Mus musculus
scientific_name
cytosine-phosphate-guanine dinucleotides; polyinosinic:polycytidylic acid
stimulus
C57BL/6
strain
Sample 21
title