GSM6208364: WT Leucine deficient 2; Mus musculus; OTHER
2 μg of RNA was separated and excised from a 15% urea-polyacrylamide gel. From the gel slices, RNA was extracted overnight at 4°C on a rotating wheel before precipitation in isopropanol during 3h at -20°C. RNA3'-end repair was carried out with 2 U/μl of T4 PNK (Lucigen) prior to a 2h adaptor ligation at 25°C using 1.5 μl of ligase mix (3.3 U/μL of T4 RNA Ligase 1 (NEB), 66.6 U/μl of T4 RNA Ligase 2 Deletion Mutant (Lucigen) and 13.3 U/μL of riboguard (Lucigen)) and 1 uL of a 20 μM 5'-adenylated DNA adaptor (containing sample barcode and a 8N unique molecular identifiers (UMIs)). Adaptor removal was carried out by treating individual libraries with 2.7 U/μl of 5'deadenylase (NEB) and 5-5 U/μL of RecJ exonuclease (Lucigen) for 1h at 30°C and 1h at 37°C. Using Zymo Clean & Concentrator columns, samples were purified and pooled . Ribosomal RNA was depleted according to siTools Biotech rRNA depletion kit specifications with minor variations: 4 μl of a custom-made riboPOOL was used to deplete rRNA and the clean-up step was done using Zymo Clean & Concentrator columns