GSM2544708: K562, dCas9-KRAB, infected with sgRNA64 (rep4); treated with TNFalpha for 1h; Homo sapiens; RNA-Seq
total RNA was extracted from cells using Trizol Reagent (Thermo), and were subjected to enrichment of polyA+ RNA using the NEBNext Poly(A) kit (NEB). Strand-specific polyA+ RNA-Seq was performed using the LM-Seq method, as described previously. Briefly, PolyA+ RNA was fragmented at 85°C in SmartScribe first-strand buffer (CloneTech) supplemented with a 3’ sequencing adapter containing a random hexamer sequence.  The fragmented RNA were subjected to first strand cDNA synthesis, followed by RNase A/H treatment.  The remaining single-stranded DNA were ligated to a 5’ sequencing adapter.  The libraries were then amplified by PCR using Illumina TruSeq-compatible primers and purified using SPRI purification beads.