Curated Sample Data

Antigen Class
Input control
Input control
Cell type Class
Cell type
0-12h embryos

Cell type information


Attributes by Original Data Submitter

0-12 hour old embryos
developmental stage
0-12 hour old embryos
Oregon R
chip antibody
chromatin batch number

Metadata from Sequence Read Archive

Library Description

Un-staged embryos were dechorinated using 120 ml final, 1:5 diluted, sodium hypochloride for 3 min. The embryos were thoroughly washed and fixed in the fixing solution [30 ml n-Heptane solution and 10 ml fixing buffer containing 0.1 M NaCl, 0.05 M HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 3.7% Formaldehyde for 15 min at 16-18°C on a rotating wheel. Fixation was then quenched by adding 125 mM, final, Glycine to the tube. The embryos were subsequently washed using PBS [+ 0.01% Triton-X100] for 10 min and stored at -80°C until further use. For nuclei isolation, embryos were slowly thawed and dounced using glass homogenizer A and B (20 strokes each) in ice-cold NX-I buffer [15 mM HEPES pH 7.6, 10 mM KCl, 2 mM MgCl2, 0.5 mM EGTA, 0.1 mM EDTA, 350 mM sucrose, 1 mM DTT, 0.2 mM PMSF, Protease inhibitors Leupeptin, Pepstatin and Aprotinin (10 µg/ml)]. The lysate was filtered through Miracloth and nuclei were spun down at 3500 rpm, 10 min, 4°C. The pellet (brown) was washed in NX-I buffer. Finally, the nuclei were resuspended in RIPA [1% Triton X-100, 0.1% Sodium deoxycholate, 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.1% SDS, 1 mM PMSF] and washed 3 times. Nuclei were then counted and aliquoted to ~109 nuclei/ml. Fractions can be stored at -80°C until shearing. For shearing and ChIP, thawed nuclei were adjusted to ~2x108 in RIPA and sheared in Covaris S220 system (Covaris Inc. MA, USA) at power 110 Watts, duty factor 20%, and cycles per burst 200 for 25 min. Chromatin was pre-cleared using protein A+G beads (1:1) mix for 1h, 4°C. 200 µl of chromatin was taken for chromatin immunoprecipitation in 500 µl reaction. The ChIP antibodies are all custom antibodies produced in our lab. 4 µl of antibodies (Rb1-ACF1, Rb2-ACF1, Rb-RSF1) were incubated per reaction overnight at 4°C. RIPA equilibrated protein A+G (1:1) mix was then added to pull down the immune-complexes for 3h, 4°C. For Rat monoclonal antibody, the chromatin immune-precipitation step was performed using presorbed protein G beads, in an excess of antibody, for 3hr at 4°C. Beads were washed subsequently 5 times in 1 ml RIPA buffer. Residual RNA was digested by RNase (10 µg) at 37°C for 20 min. Subsequent protein digestion (25 µg Proteinase K) and reversal of cross-linking were performed together at 68°C for 2 hrs. DNA was purified using GenElute™ PCR Clean-Up Kit (Sigma, NA1020). ChIP DNA was quantified using Qubit® dsDNA HS Assay Kit (Life technologies, and sequencing libraries were prepared using MicroPlex Library Preparation™ kit (Diagenode, Cat. No. C05010011) by taking 2 ng of starting material whenever possible. PCR amplification was monitored by quantifying amplified libraries (maximum 19 cycles).

Platform Information

Illumina HiSeq 1500

External Database Query

Logs in read processing pipeline

Number of total reads
Reads aligned (%)
Duplicates removed (%)
Number of peaks
1695 (qval < 1E-05)

Sequence Quality Data from DBCLS SRA