title: GSM6923451: hAFE_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 3-5 million cells were used for each histone ChIPseq experiment. Cells were cross-linked with 1% formaldehyde for 10 minutes, followed by quenching with 125mM glycine for 4-5 minutes and washed for twice with PBS. The cell pellet was lysed in cell lysis buffer (20mM Tris-HCl, pH8, 85mM KCl, 0.5% NP-40) supplemented with 1x protease inhibitor cocktail (Roche, 11836170001) on ice for 20 minutes then pelleted at 5000rpm for 10 minutes. The pellet was resuspended in sonication buffer (10mM Tris pH7.5, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) with 1x protease inhibitor cocktail and incubated for 10 minutes at 4oC. In order to achieve 200-700nt DNA fragmentation range, nuclei were sonicated (Bronson sonifier model 250 with the following conditions: Amplitude = 15%, Time interval = 3 min (total of 8-12min) and pulse ON/OFF = 0.7sec/1.3sec). Chromatin was pre-cleared with Dynabeads Protein A (Invitrogen, 10002D) for 1 hour and incubated with antibody overnight at 4C. On the following day, 30-40ul of Dynabeads Protein A were added to the chromatin and antibody mix for 2-3 hours and then the captured immuno-complexes were washed as follows – 1x low-salt buffer, 1x High-salt buffer, 1x LiCl salt buffer and 1x TE. And the washed immuno-complexes were eluted in ChIP-DNA elution buffer (10mM TriHcl pH8, 100mM NaCl, 20mM EDTA and 1% SDS) for 20 minutes, reverse cross-linked overnight at 65C, followed by proteinase K treatment (Invitrogen, 25530-049), RNase A (Thermo Fischer Scientific, ENO531) treatment and Phenol:Chloroform:isoamyl alcohol extraction. The illumina library construction steps were carried out with 5-10ng of purified ChIP'ed DNA. During the library construction, PCR purification was performed after every step using Qiagen kit (QIAquick PCR purification 28104 or QIAquick gel extraction 28706). The library reaction steps with ChIP'ed DNA as follows: 1.) End-repair - T4 PNK (NEB, M0201L), Klenow fragment (NEB, M0210S), T4 DNA Polymerase (NEB, M0203L) and dNTP mix (Thermo Fischer Scientific, 18427013) incubated at RT for 45min, 2.) 3' end A-base addition - 3' -> 5' exo- Klenow fragment (NEB, M0212L) and dATP (NEB, N0440S) incubated at 37oC for 30min, 3.) Adapter ligation - T4 DNA ligase (NEB, M0202M) and illumina adapters incubated at 16C overnight and 4.) PCR amplification - 2x PfU Ultra Hotstart master mix (Agilent, 600850-51) for 10-14 cycles. The amplified libraries were size selected for 200-450nt on 2% Agarose E-gel (Thermo Fischer Scientific, G402002). 