title: GSM6971596: HBEC3KT cells, transduced with dCAS9-VP64, P65, and sgRNA targeting SCREEM2 promoter, replicate 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using RLT buffer supplemented with Beta-mercaptoethanol according to the manufacturer's instructions (Qiagen). Isolated RNA was quantified by spectrophotometry, and RNA concentrations were normalized. Ribo-depletion-RNA-seq libraries were prepared with KAPA RNA Hyperprep kit with Riboerase (Roche) according to manufacturer's instruction. RNA samples were normalized to 300 ngs input in total 10ul and 0.6ul of 1:100 dilution ERCC (mix 1) (Thermo Fisher) was spiked in to each sample. First, ribosomal RNA were depleted using RiboErase. Purified RNA was then fragmented at 85°C for 6 mins, targeting fragments range 250-300bp. Fragmented RNA is reverse transcribed with an incubation of 25°C for 10mins, 42°C for 15mins and an inactivation step at 70C for 15mins. This was followed by second strand synthesis and A-tailing at 16C for 30mins and 62C for 10 min. A-tailed, double stranded cDNA fragments were ligated with illumina unique adaptors (Illumina). Adaptor-ligated DNA was purified using Ampure XP beads. This is followed by 10 cycles of PCR amplification. The final library was cleaned up using AMpure XP beads. Quantification of libraries were performed using real-time qPCR (Thermo Fisher). Sequencing was performed on Illumina NovaSeq platform on an S1 flowcell generating paired end reads of 150bp.