title: GSM2054781: Input_RPB7_Rep_2; Tetrahymena thermophila CU428; ChIP-Seq
library_construction_protocol: For native ChIP, 2x108 vegetative growing Tetrahymena cells of RPB7-CHA were collected at 350 g for 5 minutes. After cell lysis, nuclei were isolated by differential centrifugation following established protocols. MAC fractions containing less than 5% MIC (count/count) were used for MNase digestion. MNase digestion was carried out at room temperature for 15 min (50 mM Tris-HCl, 5 mM CaCl2, 1 mM β-mercaptoethanol, 0.1% NP-40, 0.1 mg/ml BSA, 2 U/μL MNase), stopped by adding 10 mM EGTA, 1 mM EDTA and 10 mM sodium butyric. Add equal volume of T350+Tx+Pi buffer (350 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) with PMSF and RNase A. After centrifuge, the supernatant was collected as input fraction. Anti-HA Magnetic Beads (Mouse) (Pierce #88836) was added to the input and incubated at 4oC with rotating for overnight. After wash with T150+Tx+Pi buffer (150 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) (three times) and T350+Tx+Pi buffer (350 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) (one time), beads were eluted with 500 μL of HA peptide and DNA was recovered according to standard protocol. Recovered DNA product was sent to DNA sequencing core for CHIP-seq. For crosslink ChIP, 1x108 vegetative growing WT Tetrahymena cells were resuspended in 50 mL of fixation buffer (1% paraformaldehyde in 1x DPBS) and incubated at room temperature for 30 minutes. Cells were washed with ice-cold 1x DPBS for three times. Add 5 mL of SDS Lysis buffer and PMSF to the cell pellet and homogenize cells fully with Syringe passage followed by sonication (30s on, 30s off for 20 minutes as a cycle, five cycles in total). After centrifuge and filtration, the soluble fraction was recovered and diluted in the CHIP dilution buffer (1:5) as the input. 10 μg of α-H3K4me2 antibody (abcam 07-030) was added into the input fraction and incubated at 4oC overnight. On the second day, 50 μL of pre-washed Dynal magnetic beads was added into the input-antibody mixture and incubated at 4oC with rotating for 2 hours. After washing with Wash Buffer A (three times), RIPA Wash Buffer (three times), Last Wash Buffer (two times). Beads were eluted with 500 μL of SDS Elution Buffer and DNA was recovered according to standard protocol. Recovered DNA product was sent to DNA sequencing core for CHIP-seq. Libraries were prepared with the Illumina TruSeq protocol according to the manufacturer’s protocol.
attributes.tag: GEO Accession
attributes.value: GSM2054781