title: GSM5583678: ChIL-H3K27ac - CTX injurured TA - Day 3 - rep. 3; Mus musculus; ChIP-Seq
library_construction_protocol: Each cryo-tissue section (10 um) was placed onto u-plate 96 well bottom (Ibidi #89626) and the plate was stored at -80ºC freezer without backing to room temperature until use. Tissue section was fixed with 4%PFA in 0.3% TritonX-100/PBS for five minutes and washed with 0.3% TritonX-100/PBS. Double blocking were performed using blocking one (Nacalai #03953) and M.O.M blocking reagent (Vector Laboratories #BMK-2202) as instructed. The sections were incubated with primary antibody in M.O.M. protein concentrate/PBS at 4ºC overnight, followed by incubating with ChIL probe at same condition but in addition of NaCl at 0.5 M concentration. ChIL-seq was performed according to ChIL (Harada et al., 2018) with some modifications. Longer incubation time is used for some steps (Extended Tn5 binding 1hr and fill-in step 2hrs),  Thermo T7 RNA polymerase (100 U per well; Toyobo), and 15 cycles of PCR amplification. Column purification (Zymo #D4013) and x0.5 volume of AMpure beads (Beckman Coulter) selection were performed to obtain 200-500 bp average of library.  For RNA-seq, total RNA was extracted from the cryo sections (up to five serial sections) using Direct-Zol kit (Zymo #R2063). The total of 10 ng RNA was used for library preparation using SMART-Seq Stranded Kit (Takara) as instructed.