GSM4979923: ChIP-seq_PAX8_786-M1A_2; Homo sapiens; ChIP-Seq
Approximately 30mg of tumor tissue was homogenized in the Precellys (Bertin instruments) for 30sec and crosslinked with 1% formaldehyde-supplemented media for 10 minutes. The reaction was quenched with 0.125M glycine for 5 minutes and followed by PBS washes twice. The cells were either pelleted and stored at -80oC or subjected to immunoprecipitation. For IP, the protein A/G magnetic beads (Thermo, 26162) were first equilibrated by washing the beads with 0.5% BSA in PBS three times. The beads were then incubated with antibodies in 0.5% BSA in PBS at 4oC while rotating for a minimum of 4 hours. The crosslinked cells were resuspended and dounced in lysis buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2mM EDTA pH 8.0, 0.1% SDS and 1% Triton X-100), followed by sonication in the Bioruptor (Diagenode) for 14 cycles, 30” on / 30” off. The lysates were spun down at 4oC for 20 minutes at 14000 rpm. The supernatants were added onto the antibody-conjugated magnetic beads and incubated overnight at 4oC while rotating. On the following day, the beads were washed three times with low salt buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton) and high salt buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1% Triton) once. The DNA bound to the antibody-conjugated beads was eluted with elution buffer (50 mM NaHCO3, 1% SDS) and de-crosslinked by shaking at 1000 rpm for 3 hours at 65oC. De-crosslinked DNA was purified using the QuickClean II PCR Extraction Kit (Genescript L00419-100) according to the manufacturer's recommendations. Purified ChIP DNA was subjected to Illumina sequencing. Sequencing libraries were prepared by using the KAPA Hyper Prep Kit (KR0961) according to the manufacturer's recommendations. Adapter-ligated libraries were size-selected using Agencourt AmPure XP beads (Beckman Coulter A63880) to obtain fragments of 150-350bp. Size-selected fragments were amplified for 15 cycles using the KAPA HiFi HotStart Ready mix and the amplified libraries were pooled in equimolar concentration for Illumina sequencing on a HiSeq4000 instrument.