GSM8488510: SPT6 OFF 48 h Rep2 RNA-seq; Mus musculus; RNA-Seq
Cells were collected in TRIzol (ThermoFisher) at 10 million cells/mL TRIzol. RNA was extracted with the Direct-zol RNA miniprep plus kit (R2072, Zymo Research) by adding an equal volume of 100% ethanol to TRIzol collection. Mixture was then added to columns from the kit. Bound RNA was washed with Wash Buffer and incubated for 15 min on the column with DNase I. Bound RNA was then washed with Pre-wash Buffer and Wash Buffer. Columns were dried by centrifugation at >15,000 x g and RNA was eluted with 50 µL pure water. The quantity and purity of collected RNA was checked with a NanoDrop spectrophotometer. RNA quality was assessed using an Agilent 2100 Bioanalyzer or TapeStation to confirm RNA integrity (RIN) values were above 9. RNA-seq libraries were prepared with the NEBNext rRNA Depletion Kit v2 and NEBNext Ultra II Directional RNA Library Prep kit (E7405L, E7765S, NEB) using unique dual indices (E6440, NEB). 1 µg total RNA was input for each library. RNA was hybridized with rRNA probes by denaturation for 2 min at 95 °C and gradual temperature decrease (-0.1 °C/s) to 22 °C. RNase H digestion was then performed followed by DNase I digestion. rRNA and DNA depleted total RNA was purified and then fragmented and primed with random primers. Double-stranded cDNA was synthesized, purified, and ends were repaired and prepared with A-tailing. Adaptors were diluted according to RNA input quantity (1:5 for 1 µg total RNA input). cDNA and adapters were ligated followed by USER Enzyme digestion. The ligation mix was purified and eluted with 17 µL 0.1x TE. Purified ligations were then enriched with PCR using unique dual index primers and 7 amplification cycles. Amplified libraries were quantified with Qubit and analyzed with an Agilent 2100 Bioanalyzer or Agilent 4150 TapeStation. The molecular weights of library fragments within 200-700 bp and their percentages of total were calculated with Bioanalyzer or TapeStation results.