GSM1985861: NHF1 (6-4)PP 1h XR-seq Rep1; Homo sapiens; OTHER
Cell pellets from one 150mm tissue culture dish were resuspended in 400 µl of ice cold Buffer A (25 mM Hepes pH 7.9, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 12.5% glycerol, 0.5% NP-40) and incubated for 10 min on ice. Resuspended cells were transferred to an ice-cold Dounce homogenizer/tissue grinder and lysed on ice with 15 strokes using a tight plunger. The chromatin fraction was then pelleted by centrifugation for 30 min at 16, 873 x g at 4oC. The supernatants were collected, 2 µg anti-p89 (XPB, Santa Cruz sc293 lot L1514), 1 µg anti-p62 (Santa Cruz sc292  lot F2113) and 200 µg RNaseA (Sigma R4642) were added. The reactions were rotated for 3-5 hrs at 4oC and then incubated overnight with 15 µL of recombinant protein A/G PLUS-agarose (Santa Cruz sc2003). After washing three times with 1 ml of buffer A and once with 1ml of buffer B (25 mM Hepes pH 7.9, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 12.5% glycerol, 1% NP-40), DNA was eluted from immunoprecipitates with 100 µl of buffer C (10 mM Tris-Cl pH 7.5, 1 mM EDTA, and 1% SDS) at 65oC for 15 min. The eluted DNA was then isolated by phenol/chloroform extraction followed by ethanol precipitation. DNA pellets were resuspended in 45 µl water, and then incubated with 5 µL of RNaseA/T1 mixture (Thermo EN0551) for 1 hr at 37oC. After phenol-chloroform extraction, purification through G50 filtration columns (GE Healthcare) and ethanol precipitation, the DNA was then used for ligation.  To add double-stranded adaptors to both ends, purified excised oligomers were incubated with 40 pmol 5′ adaptor and  100 pmol 3′ adaptor in 10 µL 2x Hybridization Buffer (20mM Tris-HCl pH7.5, 200mM NaCl, 0.2mM EDTA) at 60oC for 10min, then 16oC for 5min in a thermal cycler. To perform ligation, 4 µL 5x ligase buffer, 1 µL T4 DNA ligase HC (Life 15224-041), 1 µL 50% PEG8000 (NEB) and 4 µL H2O were added to each reaction. The reactions were incubated at 16oC overnight. After phenol-chloroform extraction and ethanol precipitation, ligation products were subjected to immunoprecipitation with anti-CPD (For the 16h sample - Cosmo NM-DND-001 lot TM-C-08, for all the rest, Kamiya cat MC-062, lot 11569062) or anti-(6-4)PP (Cosmo cat NM-DND-002, lot TM-P-04) antibodies.Purified DNA were repaired by (6-4)PP photolyase or CPD photolyase. DNA containing (6-4)PP or CPD were incubated with 1.25pmol E.coli (6-4)PP photolyase or 20pmol D.melanogaster CPD photolyase in 50 µL PLR buffer for 2hr on ice and irradiated with 18mW/cm2 of 366 nm light. 1% of unrepaired or repaired samples were used for quality check. After phenol-chloroform extraction and ethanol precipitation, repaired DNA were PCR amplified by Kapa Hifi HotStart ReadyMix with RP1 and RPIn (n means different index sequences, compatible with Illumina TrueSeq Small RNA Kit.) The PCR products were extracted with phenol-chloroform, precipitated with ethanol and subjected to electrophoresis in a 10% native polyacrylamide gel in 1xTBE. Gel slices corresponding to 130 to 155-nt fragments were excised. The DNA was eluted for 6 hrs in 0.3 M NaCl at room temperature, precipitated with ethanol and resuspended in buffer EB (10mM Tris-HCl pH8.5).