GSM5050066: Rnmt cKO 4 CMC; Mus musculus; ncRNA-Seq
Total RNA was extracted using trizol. Libraries were made following the pseudoruridine sequencing protocol published by Schwartz, S. et al 2014. 300ng of total RNA from each replicate was made up in BEU buffer (50mM bicine pH 8.3, 4mM EDTA, 7M urea) for the mock treated sample or BEU buffer plus 0.17M CMC (N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) (Sigma). Samples were heated to 37°C for 20 mins, then RNA precipitated by adding 100ul buffer A (0.3M NaOAc, 0.1mM EDTA), 1 ul glycogen and 700ul ethanol and washed 1X in 75% EtOH. Samples were resuspended in buffer A, then precipitated as before with ethanol to remove any remaining CMC. RNA was made up in 50mM sodium bicarbonate pH10.4 and heated to 37°C for 3 hours (this removes unwanted CMC adducts). RNA was precipitated with 100ul buffer A, 1ul glycogen, 700ul Ethanol, and washed in 75% ethanol. RNA was made up in 50mM sodium bicarbonate pH9.2 and heated to 95C for 8 mins to fragment, then onto ice. RNA was precipitated with 200ul buffer A, 1 ul glycogen, 500ul ethanol, and washed in 75% ethanol. RNA was dephosphorylated with FastAP (thermo), and linker ligated with high concentration T4 ligase (NEB). Products were reverse transcribed, then the second linker ligated using the high concentration T4 ligase (NEB). Clean ups between these steps were conducted using myOne silane beads. The libraries were PCR amplified and gel purified, then sequenced with a Nextseq High Output v2.5 kit (75 cycles, single end).