GSM8223693: mESC, naive pluripotency, TKO, shLBR, day1, rep3; Mus musculus; RNA-Seq
Total RNA was isolated from cells using the RNeasy Plus Mini Kit (QIAGEN 74136). RNA was treated with DNAse using the TURBO DNA-free kit (Invitrogen™ AM1907) prior to prepping libraries. RNA quality was assessed by electrophoresis on a 1% agarose gel and detecting 28S and 18S rRNA bands with ethidium bromide staining (0.6µg/mL). A version of the Tecan Genomics' Universal Plus mRNA prep kit (Tecan Genomics 0520B-A01) was modified for rRNA depletion by replacing the mRNA isolation in the first segment of the protocol with ribo depletion using FastSelect rRNA (Qiagen 334387) as follows: 200ng of total RNA in 10µL of water was mixed with 10µL of Universal Plus 2X Fragmentation Buffer and 1µL FastSelect. The solutions were fragmented and ribo depleted simultaneously per the FastSelect protocol. 20µL of the resulting fragmented/ribo depleted RNA were prepped per the Universal Plus protocol starting with First Strand Synthesis. The Universal Plus Unique Dual Index Set B was used, and the samples were quality checked using a MiniSeq benchtop sequencer (Illumina).