GSM2627258: CD4_resting_FOSL_ChIP-seq_rep; Homo sapiens; ChIP-Seq
The anti-FOSL1 antibody (Santa Cruz Biotechnoloy, Inc. The Cat# is sc-183)-immunoprecipetated chromatin on beads were added with 100ul EB buffer, 3 ul  of 10% SDS and 5 ul  of 20 mg/ml proteinase K. Incubate at 65C overnight. Extract 1x with 200 ul  of phenol/chloroform (PCI) for DNA. Add 1 ul  of 20 mg/ml glycogen, final 0.2M NaCl and final 70% Ethanol to precipitate. Incubate at -80°C for 1 hour. Spin at max speed for 30 min. Wash pellet once with 1 ml 70% ethanol, then spin for 10 min. at 4°C. Air dry and resuspend ChIP DNA in 40 ul  1xTE. ChIP DNA is subjected to DN Aend repair using the Epicentre DNA END-Repair kit (Cat. No. ER0720, Epicentre Biotechnologies) according to the manufacturer instruction. Repair DNA was cleaned with the MinElute column using MinElute Reaction Cleanup protocol (Qiagen, pgs 28-29 of MinElute Handbook) and eluted with 40ul EB. Add 5 ul  10x NEB Buffer #2. 1 ul  10 mM dATP and 3 ul  5 U/ul  Klenow Fragment (3´→5´ exo–). Incubate for 30 min at 37°C. Purify the reaction again as above and elute the DNA in 22 ul  of EB. Add 3 μl 10x T4 DNA ligase buffer, 2 μl adapter (15uM,  annealed from primer: multiplexing    adapter-top with multiplexing adapter-bottom that is the same as Illumina's InPE adaptor), mix then add 3 μl T4 DNA ligase (400 units/μl). Incubate for 30 min at room temperature. Add 6 ul H2O and load 18ul per lane on E-gel. Run 10 min. Gel purify the fragments from 200-400 bp. Elute DNA in 22ul EB. Add 25 μl of master mix (HF fusion), 1 μl of 10uM Multiplexing PCR Primer 1.0, 1 μl of 10uM one of the indexed primer2: PCR Primer2- IndexM1-64, or PCR Primer2- IndexM2-64, till PCR Primer2- IndexM12-64 (M stands for Modified, -64 means it is 64bp long). PCR Conditions:   98°C for 30 sec. 17 cycles of 98°C, 10”; 65°C, 30”; 72°C, 30”, 72°C for 5 min. 4°C hold. Gel purify the 250-450 bp fragments for sequencing.