Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
CTCF

Cell type

Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
HeLa-S3
cell population
synchronized
tsa treated
no
cell line
HeLa-S3
chip antibody
anti CTCF G.758.4 (MA5-11187, Invitrogen)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP was performed either manually on individual samples or automatically using the Bravo liquid handling platform (Agilent model 16050-102, “Bravo”) as described previously (Busby et al. 2016). Illumina Library construction reactions were performed as we described previously (Garber & Yosef et. al. 2012). SPRI (Solid-phase reversible immobilization; AMPure XP beads (Agencourt)) cleanup steps were done using the Bravo liquid handling platform (Agilent) and a modified version of (Fisher et al., 2011). After the final ChIP step, 120µl SPRI was added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant was separated from the beads using a 96-well magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. DNA was eluted in 40µl EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing (25 times). The remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) the SPRI cleanup involves addition of PEG buffer (20% PEG and 2.5 M NaCl) to the reaction products. Next, plates were transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are washed on the magnet with 150µl 70% ethanol and air dried for 4 minutes. The DNA is eluted with 40µl of EB buffer (mixing 25 times). Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27µl of a master mix (17µl master mix (5µl T4 buffer, 5µl BSA-1mg/ml, 5µl ATP-10mM -2µl dNTPs 10 mM)) plus 5µl T4 PNK enzyme, 5µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12oC for 15 min, 25 oC for 15 min, and finally cooled to 4 oC. The SPRI bead clean up method was used to purify the product (147µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40µl EB). The A-base addition was performed by adding 20µl master mix (17µl A-base add mix, 3µl Klenow to each well and incubated at 37oC for 30 min in a thermal cycler. Product was purified using 132µl PEG buffer and eluted in 19µl EB. Adaptor ligation: was performed by adding 34µl of a master mix (29µl 2x DNA ligase buffer, 5µl DNA ligase) and 5µl indexed oligo adaptors (0.75 uM ) followed by 15min incubation in 25oC in a thermal cycler. Next, ligated products were purified using SPRI bead clean up with size selection (15.5µl of PEG buffer) and eluted in 40µl EB. The final step of PCR enrichment was was performed by adding 10µl of a master mix (2µl Forward/Reverse Index Primer, 0.5µl dNTP mix, 5µl 10x PfuUltra Buffer, 1µl PfuUltra-II Fusion, 1.5µl Nuclease free water) to each well. Using a thermal cycler, programed to 95C for 2 min, followed by 16 cycles of (95oC for 30 sec, 55oC for 30 sec, 72oC for 60 sec), and a final incubation at 72oC for 10 min. The last SPRI clean up was coupled to size selection (35µl SPRI beads were added to each sample and eluted in 40µl).

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
46395850
Reads aligned (%)
165.6
Duplicates removed (%)
13.1
Number of peaks
29031 (qval < 1E-05)

hg38

Number of total reads
46395850
Reads aligned (%)
167.6
Duplicates removed (%)
12.7
Number of peaks
28926 (qval < 1E-05)

Base call quality data from DBCLS SRA