GSM2573003: 18S SSIII 0.5% DMS A; Mus musculus; OTHER
DMS-treated and untreated cells were collected in PBS by centrifugation (3 min, 1000 g, 4ᵒC). Then cells were lysed with a dounce homogenizer 15 times in 15 mM Tris pH 7.4, 60 mM KCl, 2 mM EDTA. The cells were collected by centrifugation (3 min, 1000 g), and the supernatant was discarded. The remaining pellet was resuspended in TRIzol LS (Life Technologies) and RNA was extracted according to the manufacturer’s instructions. Extracted RNA was treated with RQ1 DNase (Promega) for 1 hr at 37ᵒC, extracted with phenol/chloroform, and precipitated with ethanol. For duplicate treated and untreated samples, RNA (1 μg) was brought to 12 μl with 10 pmol total of primers (primer sets were identical to those in Fang et al. 2015) The primer/RNA mix was brought to 70ᵒC for 5 min, then 4ᵒC for 2 min to anneal primers. Then 3 μl 5x reverse transcription buffer was added and mixed before incubating samples at 55ᵒC for 10 min. Then 5 μl RT mix was added  and reactions were incubated for 45 min at 55ᵒC for SSIII, 42ᵒC for AMV, or 60ᵒC for TGIRT-II. After reverse transcription, reactions were treated with 1 μl RNase A and 0.5 μl RNase H for 1 hr at 37ᵒC. Reverse transcription reaction cDNA products were purified with AMPure XP beads by adding 1 volume beads solution (at room temperature), 3 volumes PEG solution (2.5 M NaCl, 20% PEG 8000), and 1 volume iPrOH, mixed and allowed to sit at room temperature for 15 min. Beads were immobilized for 10 min on a magnetic rack, then washed twice with fresh 80% EtOH, allowed to dry for 10 minutes, then eluted in 16 μl dH2O. Ligation of 3’ adaptor was performed, and two subsequent rounds of PCR amplification, with Illumina indexes added in the second round.