GSM8883053: invitro MCF7 100Q tRF SC rep2 Input; Homo sapiens; RNA-Seq
Total RNA was extracted from input and polysome fractions using the TRIzol reagent (15596018, ThermoFisher) and MSR-seq libraries were built as previously described. Briefly, 3 volumes of TRIzol reagent were added to input lysate or pooled polysome fractions for each sample. Total RNA was then extracted by following the manufacturer's manual. 20 µg total RNA was used to extract polyA+ RNA using PolyATtract mRNA Isolation Systems (Z5310, Promega) by following the manufacturer's manual. 400ng polyA+ RNA was fragmented at 94°C for 3 min (~300nt) using NEBNext Magnesium RNA Fragmentation Module (E6150S, NEB). Fragmented RNA mixture was then diluted to 50 µl using sterile H2O and purified using RNA Clean & Concentrator-5 (R1016, Zymo). RNA was eluted in 8 μl sterile H2O and 1 μl T4 PNK buffer and 1 µl 10 U/μl T4 PNK (EK0031, ThermoFisher) was added to each sample and incubated at 37°C for 30 min to repair the RNA ends followed by inactivation at 75°C for 10 minutes. The end-repaired fragmented RNA (10 µl) was used as the input RNA for first adaptor ligation in MSR-seq. Standard MSR-seq procedure was followed afterwards. The PCR amplified MSR-seq libraries were purified using AMPure XP (A63880, Beckman Coulter) beads or gel. MSR-seq (PMID 35513407)