GSM4669087: ChIRP_LINE1.Even; Mus musculus; OTHER
ChIRP was performed as previously described with some modifications2,3. In brief, a set of biotinylated antisense oligos with 59-nt long probes tiled along the L1 consensus sequence (6.5 kb) or Malat1 RNA, and 20-nt probes tiled along the IAP consensus sequence or LacZ. Label probes based on their positions along the RNA, and separate them into 'even' and 'odd' pools, to create two independent probe sets for ChIRP experiments. Ythdc1 cKO ESCs were treated with DMSO or 4OHT (1 μg/ml) for 4 days, then cells were harvested by 0.25% trypsin digestion and cross-linked with 2 mM DSP (Thermo Scientific, # 22586) in PBS at room temperature for 30 min with gentle end to end rotation. The cells were further fixed in suspension for 10 min in 3.7% formaldehyde, and quenched with 250 mM Glycine for 5 minutes at room temperature. Cells were then washed with ice-cold PBS for three times and snap frozen in liquid nitrogen. 1 × 107 cells were resuspended with 500 μl DNase I digestion solution (20mM Tris-HCl, pH7.5, 5mM MgCl2, 0.5mM CaCl2, 0.5% Triton X-100) with 1/20 volume of Ribonucleoside Vanadyl Complex (RVC, NEB, #S1402S), 5 μl protease inhibitor cocktail (Roche, #4693132001), 5 μl 100mM PMSF and 12 U/ml Dnase I (Invitrogen, #AM2222); the reaction was rotated for 10 min at 37°C and stopped with 20 mM EDTA. After centrifugation, chromatin was resuspended in nuclear lysis buffer (50mM Tris-HCl, pH7.5, 10mM EDTA, 1% SDS, inhibitors) and sonicated with Bioruptor Pico (5 cycles, 30s on, 30s off, Diagenode). The supernatant was collected for ChIRP experiments after centrifuged at 14,000 ×g for 10 min at 4°C and save some lysate as 'input' samples. For the Rnase treatment control, samples were treated with 10 μg/ml Rnase A/T1 at 37°C for 20 min. For hybridization, samples were incubated with ChIRP probes (1 μl of 100 μM probes per 1 ml of lysate), supplemented with one-fourth volume of 5 x hybridization buffer (50 mM Tris-HCl, pH7.5, 10 mM EDTA, 1.5 M NaCl, 50% formamide). After rotated at 39°C for 3h, 50 μl prebalanced streptavidin M280 beads were then added into the hybridization and incubated for an additional 3h. The beads were washed 5 times with 0.2 × SSC wash buffer with 1% SDS at 42°C. To elute, the beads were washed once with SDS elution buffer (50 mM Tris-HCl, 5 mM MgCl2, 75 mM NaCl, 1% SDS) at 39°C for 20 min, and once with elution buffer (50 mM Tris-HCl, 5 mM MgCl2, 75mM NaCl, 0.1% Triton X-100) at 39°C for 5 min. DNA was eluted from the beads by RNase H treatment in two sequential incubations with Rnase H (NEB, #M0297L) at 37°C for 20 min, and with SDS elution buffer at room temperature for 2 min, combining all eluents. Crosslinking was reversed by treatment with 0.1 μg /μl protease K (NEB, #P8107S), 150 mM NaCl, and 10 mM EDTA at 65°C overnight and the DNA was purified using the MinElute PCR Purification Kit (Qiagen, #28006). ChIRP enrichments were analyzed by qPCR of the purified DNA. The libraries were constructed by a KAPA HTP/LTP Library Preparation Kit (#KK8234) according to the manufacturer's instructions.
GEO Accession
GSM4669087