title: GSM3579126: MIC 3 hpm Hi-C; Tetrahymena thermophila; Hi-C
library_construction_protocol: We crosslinked conjugated Tetrahymena theromphila cells at 1.5 hpm, 3 hpm and 24 hpm (hpm: hours post-mixing) with 1% formaldehyde for 10 minutes at room temperature. For Hi-C, we followed in-situ Hi-C protocol, the Tetrahymena cells were lysed. Then the lysed cells, isolated MIC or MAC were digested by MboI or HindIII overnight at 37℃. DNA fragment ends were filled and labeled with dCTP, dGTP, dTTP and biotin-dATP. Proximity ligation was performed at room temperature for 4 hours. Then the DNA were de-crosslinked at 65°C overnight with Proteinase K and DNA were purified. For HiChIP, the Tetrahymena cells were lysed. Then the lysed cells, isolated MIC or MAC were digested by MboI or HindIII overnight at 37℃. DNA fragment ends were filled and labeled with dCTP, dGTP, dTTP and biotin-dATP. Proximity ligation was performed at room temperature for 4 hours. Following the in situ ligation, we fragmented the ligated chromatin with sonication. Then the crosslinked protein/DNA was immunoprecipitated using antibodies to HA (rabbit, Cell Signaling Technology C29F4 or Abcam ab9110) and GFP (Abcam ab290) and then purified by Protein A+G immunomagnetic beads. The amount of MboI or HindIII enzyme, antibody, and beads are determined by the number of cells in each sample. After immunoprecipitation, DNA was eluted from the beads by incubating at 65°C for 4 hours with Proteinase K and purified. Both Hi-C and HiChIP DNA were sonicated and binding to M280 beads, then end-repair, dA-tailing and adapter ligation on beads. After PCR amplification and DNA purification, the libraries were sequenced on HiSeq X Ten using 150bp paired-end reads. Hi-C and HiChIP
attributes.tag: GEO Accession
attributes.value: GSM3579126