GSM7502505: HEL_AFF4KD_Pol ll (Ser-2p); Homo sapiens; ChIP-Seq
Normally, cells were fixed with 0.4%  (v/v) formaldehyde at room temperature for 10 min. To improve the ChIP efficiency, double  fixation was used. For double fixation with EGS (Thermo, cat. no. 21565) and formaldehyde, cells  were fixed initially with 1.5 mM EGS at room temperature for 30 min, and subsequently with 0.4%  formaldehyde at room temperature for 10 min. For double fixation with DMA (Thermo, cat. no.  20660) and formaldehyde, cells were fixed initially with 25 mM DMA at room temperature for 1 hr,  and subsequently with 0.4% formaldehyde at room temperature for 10 min. For sonication, fixed  cells were washed twice with PBS and resuspended in ice-cold RIPA-0.3 buffer (10 mM Tris-HCl,  1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDOC, and 0.3 M NaCl, pH 7.4) supplemented  with Protease Inhibitor Cocktail (Millipore, cat. no. 535140) at a concentration of 40 million  cells/ml; genomic DNA was disrupted to a size range of 100 to 500 bp. For immunoprecipitation,  on day 1, antibodies were diluted in RIPA-0.3 and bound to Dynabeads protein A (Thermo, cat.  no. 10002D) by incubating at 4°C for 3 hrs. Afterwards, the bead-antibody complexes were  washed twice with RIPA-0.3 and then incubated with sonicated chromatin at 4°C overnight. On  day 2, after 2 washes with RIPA-0.5, 1 wash with RIPA-0.3, 1 wash with RIPA-0, 2 washes with  LiCl buffer (10 mM Tris-HCl, 1 mM EDTA, 0.25 M LiCl, 0.25% NP-40, and 0.25% NaDOC, pH  7.4), and 2 washes with TE buffer, bound protein-DNA complexes were resuspended in elution  buffer (10 mM Tris-HCl, 1mM EDTA, 0.2 M NaCl, and 1% SDS, pH 7.4) supplemented with 10  µg/ml RNase A for both elution and RNA digestion, and incubated at 55°C for 1 hr. Then  2 Proteinase K was added to a final concentration of 200 µg/ml, and after 30 min incubation the  temperature was increased to 65°C for crosslink reversal. After incubation for 4 to 6 hrs, DNA  was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205).ChIP-seq libraries were constructed with 2-10 ng immunoprecipitated DNA. After end-repair, A_x0002_tailing, and barcode ligation, barcoded DNA was amplified by 16- to 18-cycle PCR.