GSM9267162: FKBP-SETDB1 A12A10 ES, dTAG+dox, 4sU-seq; Mus musculus; RNA-Seq
4sU labelling of RNA, purification and sequencing was as previously described with a few modifications. For 4sU labelling of newly synthesized RNA, 4sU was added to the cell culture media at a final concentration of 500mM, and cells on a nearly confluent 10cm Petri dish were incubated for 20 minutes prior to quick removal of media and direct lysis using Trizol reagent (Life Technologies). Lysates were snap frozen and kept at -80oC until further processing. RNA isolation was performed using Trizol-chloroform phase separation, followed by isopropanol precipitation of the RNA and a 75% ethanol wash prior to dissolving the RNA pellet. Purified RNA was treated using TURBO DNAse as per manufacturer's recommendation. RNA was then quantified using Nanodrop to prepare the Biotinylation reaction consisting of 7mg MTSEA biotin-XX linker (Biotium, cat. no. BT90066) dissolved in DMF at a concentration of 0.1 mg/mL, and 3.5μL of 10X Biotinylation buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA) per μg of purified RNA. This reaction was incubated at room temperature in the dark for 30 min, prior to two rounds of RNA purification using phenol:chloroform:isoamylalcohol (25:24:1) and Phase Lock Gel Heavy tubes and subsequent isopropanol precipitation with NaCl. RNA was then denatured at 65oC for 10min and immediately cooled on ice for 5 min.  Biotinylated 4sU-labelled RNA was captured using 200mL of μMACS streptavidin MicroBeads (from the μMACS Streptavidin Kit) and incubating for 15 minutes at room temperature prior to recovering using the μMacs Streptavidin Kit (Miltenyi). Samples were washed six times using Washing Buffer (100 mM Tris pH 7.5, 10 mM EDTA, 1M NaCl, 0.1% Tween20), the first three at 65oC, before elution using fresh 100 mM DTT into RLT buffer from RNeasy minElute kit which was used for a final RNA cleanup. Libraries were prepared for sequencing using the Illumina TruSeq stranded total RNA kit including the rRNA depletion. Libraries were quantified and verified using Qubit High Sensitivity DNA (Invitrogen) and the Bioanalyser High Sensitivity DNA (Agilent) kits before pooling and paired-end sequencing on an Illumina NextSeq 500 machine using NextSeq 500/550 High-Output v2.5 Kit (150 cycles) (Illumina).