Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Approximately 25*10^6 cells were cross-linked in formaldehyde (1% final concentration, 10 min at room temperature (RT)), and then quenched with glycine (5 min at RT). Fixed cells were lysed in 50mM HEPES KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4°C (Roche, 04693159001), centrifuged at 950 x g for 10 min and re-suspended in 0.2% SDS, 10mM EDTA, 140mM NaCl and 10mM Tris-HCL. Cells were then fragmented with a Branson Sonifier (model S-450D) at -4°C to a size range between 200 and 800 bp, and precipitated by centrifugation. 10 ug of antibody were pre-bound by incubating with Protein-G Dynabeads (Invitrogen100-07D) in blocking buffer (PBS supplemented with 0.5% TWEEN and 0.5% BSA) for 2 hours at room temperature. Washed beads were added to the chromatin lysate, and then incubated overnight. Samples were washed 5 times with RIPA buffer, twice with RIPA buffer supplemented with 500 mM NaCl, twice with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), once with TE (10Mm Tris-HCl pH 8.0, 1mM EDTA), and then eluted in 0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris Hcl pH 8.0 at 65°C. Eluate was incubated in 65°C for 4 hours, and then treated sequentially with RNaseA (Roche, 11119915001) for 30 min and Proteinase K (NEB, P8102S) for two hours. DNA was purified with The Agencourt AMPure XP system (Beckman Coulter Genomics, A63881). 120ul SPRI AMPure XP beads (Agencourt) were added to the reverse-crosslinked samples, pipette-mixed 15 times and incubated for 2 minutes. Supernatant were separated from the beads using a magnet for 4 minutes. Beads were washed on the magnet with 70% ethanol and then air dried for 4 minutes. The DNA was eluted in 40 ul EB buffer (10 mM Tris-HCl pH 8.0) by pipette mixing 25 times. For the remainder of the library construction process (DNA end-repair, A-base addition, adaptor ligation and enrichment) a general SPRI cleanup involves addition of buffer containing 20% PEG and 2.5 M NaCl to the DNA reaction products (without moving them from their original well position). After thorough mixing and a 2-minute incubation at room temperature, plates are transferred to a magnet plate, incubated for 4 minutes and supernatant removed. Beads are then washed on the magnet with 150ul 70% ethanol and then air dried for 4 minutes. The DNA is eluted with 40ul of EB buffer by pipette mixing 25 times. Reagent kits are prepared in advance for all enzymatic steps (New England Biolabs). The DNA end-repair was performed by adding 27 µl of a master mix (17 µl master mix (5 ul T4 buffer, 5ul BSA-1mg/ml, 5ul ATP-10mM -2ul dNTPs 10 mM), 5 ul T4 PNK enzyme, 5 µl T4 polymerase (3 units) to each well. Samples were incubated in a thermal cycler at 12C for 15 min, 25C for 15 min, and finally cooled to 4C. The SPRI bead clean up method was used to purify the product (147 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). The A-base addition was performed by adding 20 µl master mix (17 µl A-base add mix, 3 µl Klenow (3'->5' exonuclease) to each well and incubated at 37C for 30 min. in a thermal cycler. SPRI bead clean up method was used to purify the product (132 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 19 µl EB). Adaptor ligation was performed by adding 34 µl of a master mix (29 µl 2x DNA ligase buffer, 5 µl DNA ligase) to each well. Finally 5 µl PE Indexed oligo adaptors (0.75 uM ) was added to each well and samples were incubated 25C for 15 min in a thermal cycler. SPRI bead clean up with size selection was used to purify the ligated products (15.5 µl of 20% PEG, 2.5 M NaCl was added to each sample and eluted in 40 µl EB). Finally, enrichment PCR was performed by adding 10 µl of a master mix (2 µl Forward/Reverse Index Primer, 0.5 µl dNTP mix, 5 µl 10x Pfu Ultra Buffer, 1 µl Pfu Ultra-II Fusion, 1.5 µl Nuclease free water) to each well. Plate was transferred to a thermal cycler and ran a Pfu amplification program at 95C for 2 min, 16 cycles of: 95C for 30 sec, 55C for 30 sec, 72C for 60 sec, and finally 72C for 10 min. The final SPRI clean up coupled to size selection was performed (35 µl SPRI beads was added to each sample and eluted in 40 µl).