title: GSM6370616: H2BW2 Stable HeLa cell_Input1; Homo sapiens; ChIP-Seq
library_construction_protocol: The ChIP-seq from formalin-fixed paraffin-embedded testes slides is as follows. Briefly, 20 human testis sections (10 µm) were deparaffinized and rehydrated. The tissue sections were then collected and washed by 600 µl wash buffer (1x PBS, 0.5% Tween 20), followed by a 30 mins treatment with 1 µg of RNAse (Invitrogen) and three-times wash with lysis buffer (0.2x PBS, 0.1% Tween 20, 40 mM Tris-HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2). After incubation, 600 µl of lysis buffer was added to the sample, and then it was treated with 0.5 units of MNase for 5 mins at 37 ℃. The reaction was stopped by adding EDTA to the final concentration of 15 mM, and 0.06 units of Thermolabile Proteinase K (NEB) were added with incubation at 37 ℃ for 30 mins. The proteinase K reaction was stopped by incubating at 65 ℃ for 20 mins, then the samples were sonicated until the DNA fragment size became around 150-300 bp. The sample was used to perform H2BW2 IP following standard IP method with homemade anti-H2BW2 antibody (peptide synthesis based polyclonal rabbit antibody, RGSRRRHANRRGDS). To elute the DNA, 2 μl of proteinase K (NEB) was added to the sample and incubated at 65℃ overnight and DNA was extracted with a regular PCR clean-up kit. The Flag-H2BW2 stable expression HeLa cell Ch-IP experiment was performed based on ANTI-FLAG® M2 Affinity Gel based on the manufacture's instruction of Pierce™ Agarose ChIP Kit (Thermo Fisher Scientific). The DNA library was prepared following the instruction mentioned in ThruPLEX DNA-Seq Kit (Takara) with pair-end primer.