GSM3324608: Xist[4658-5090] 1500mM EDC A; Mus musculus; OTHER
After treatment, cells were lifted using Cell Scraper (Corning) and collected in PBS by centrifugation (3 min, 1000 rpm, 4 °C), and the supernatant was discarded. The RNA was extracted using TRIzol (Thermo Fisher) according to manufacturer's instructions. The extracted RNA was treated with RQ1 DNase (1 h, 37 °C; Promega), and subsequently purified with phenol/chloroform extraction and ethanol precipitation. Reverse transcription was carried out using either 2 μg (7SK) or 10 μg (Xist) of extracted RNA, and 1 pmol total of primers. The RNA/primer mix was brought up to 12 μL and incubated at 70 °C for 5 min, then 4 °C for 2 min for primer annealing. Next, 3 μL of 5X FS buffer was added for a 10 min incubation at 55 °C. Finally, 5 μL of reverse transcription mix (1 μL 5X FS buffer, 1 μL 10 mM dNTPs, 1 μL 100 mM DTT, 0.2 μL RNase OUT, 0.5 μL SuperScript III, 1.3 μL dH2O) was added and mixed before incubation at 55 °C for 45 min, followed by heat inactivation at 85 °C for 5 min. After reverse transcription, RNA was degraded by adding 0.4 μL of 5 M NaOH and incubating at 95 °C for 3 min, before neutralization with 0.4 μL of 10 M acetic acid and cooling on ice. The cDNA from reverse transcription was purified with AMPure XP beads by adding and mixing 1 volume of bead solution and 3 volumes of PEG solution (2.5 M NaCl, 20% PEG 8000) at room temperature. The mixture was let to incubate at room temperature for 15 min, before immobilizing the beads on a magnetic rack for 10 min. The beads were washed with fresh 80% ethanol twice before being dried for 10 min and eluted in 16 μL of dH2O. 3' adaptor ligation was carried out using T4 RNA Ligase I, followed by two rounds of PCR amplification, with Illumina indexes added in the second round.
GEO Accession
GSM3324608