Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
pho

Cell type

Cell type Class
Cell line
Cell type
S2
Source
Oregon R
Developmental Stage
late embryonic stage

Attributes by original data submitter

Sample

source_name
s2-cells-pho
biomaterial_provider
ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
tissue
Schneider's Drosophila Line 2 (SL2) cells
cell line
SL2
passages
10 to 12
chip antibody/selection
Pho antibody

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). 120 μg of extract were incubated in a final volume of 1 mL IP buffer (15 mM Hepes pH 7.9 / 200 mM KCl / 1.5 mM MgCl2 / 0.2 mM EDTA pH 8 / 0.25% NP-40 / 20% glycerol / 0.3 mM DTT / 1x "Complete" protease inhibitor cocktail / 1 mM PMSF) with 1:100 anti-Pho antibody (kindly provided by Judith Kassis1) for 12 hours at 4°C. 100 µL protein A/G agarose beads (Calbiochem), previously blocked with 1 mg/mL BSA, was added to each chromatin at 4°C overnight. The beads were sequentially washed trice with 5-10 volumes of 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0), PBS, and 1% SDS/PBS. Centrifugation of the beads between washing steps was carried out (7,500 rpm, 2 min). The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).

Sequencing Platform

instrument_model
Illumina MiSeq

dm3

Number of total reads
8714961
Reads aligned (%)
90.2
Duplicates removed (%)
6.0
Number of peaks
2813 (qval < 1E-05)

dm6

Number of total reads
8714961
Reads aligned (%)
89.5
Duplicates removed (%)
7.8
Number of peaks
2327 (qval < 1E-05)

Base call quality data from DBCLS SRA