title: GSM7350567: HeLa - Chip1 Subpool1 RNA Rep 1 / Chip2 Subpool3 RNA Rep 2; Homo sapiens; RNA-Seq
library_construction_protocol: 24 hours after electroporation, HEK293/HeLa cells were lysed using TRIzol (Thermo Fisher Scientific, Cat# 15596026). Each 500 ﻿μl TRIzol-lysed solution was mixed with 100 ﻿μl chloroform (Fisher Chemical, Cat# C298-500) to allow phase separation. The upper aqueous phase was transferred and mixed with equal volume ethanol (200 proof, Fisher BioReagents). The mixture was loaded to the column supplied by the Direct-zol RNA Miniprep Plus kit (Zymo Research, Cat# R2072) to isolate total RNA following the manufacturer's protocol. PolyA selection was carried out to isolate mRNA from total RNA using Dynabeads™ Oligo(dT)25 (Thermo Fisher Scientific, Cat# 61002). custom DNA-Seq and RNA-Seq The UMI-containing libraries were generated from the plasmid library (DNA) before electroporation, as well as mRNA isolated after electroporation. The mRNA was reverse transcribed into cDNA using the SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, Cat# 18090010) and a gene-specific reverse transcription (RT) primer (MPP3) that contains a 15-mer unique molecular identifier (UMI), which was synthesized as -NNNNNNNNNNNNNNN- (See Supplementary Protocol for primer sequences). After RT, mRNA was removed via RNase H treatment. Both cDNA and plasmid DNA underwent two rounds of PCR. The first-round (3 cycles) utilized custom primers (MPP2 & MPP3) to add UMIs to the cDNA or plasmid DNA samples. The low-cycle number was utilized to minimize PCR amplification bias. The first-round PCR products were cleaned up using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Next, a second-round PCR was performed using the purified first-round PCR products and primers (MPP2 & MPP4), which added sample indexes and Illumina sequencing adaptors (P5/7). To avoid over-amplification, a pilot reaction for the second-step PCR was performed using the PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific, Cat# A25742) and ran on a qPCR thermal cycler. An amplification curve was obtained for each sample to determine the cycle number before the plateau. This cycle number (or a smaller value) was then used in the second-round PCR (See Supplementary Protocol). All PCR steps for sequencing library generation were performed using the Q5 polymerase (NEB, Cat# M0492L). PCR reactions for the same sample were pooled and purified using the DNA Clean & Concentrator kit (Zymo Research, Cat# D4004). Purified PCR products were resolved on a 2% agarose gel and the band at the expected library size (377bp) was cut out and purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Cat# D4002). UMI-containing libraries made from DNA/RNA were mixed and sequenced using custom sequencing primers on Illumina Hiseq3000 PE150 or Novaseq SP PE150 with 15% PhiX spike-in.