Illumina HiSeq 4000 paired end sequencing; RNaseq of two human LUAD cell lines with knockout of STK11, KEAP1, both genes or no genes.
RNaseq of two human LUAD cell lines with knockout of STK11, KEAP1, both genes or no genes.
Cells from 24 isogenic clones (12 per cell line, 3 per genotype) were collected on the same day and at the same density from culture. Pellets were spun down at 1500xg for 5 minutes at 4 degrees C and PBS was aspirated. Pellets were frozen at -80 degrees C. RNA extraction and sequencing was done in collaboration with Genewiz. Total RNA was extracted from fresh frozen cell pellet samples using Qiagen RNeasy Plus Universal mini kit following manufacturer's instructions (Qiagen, Hilden, Germany). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA).  RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).  The sequencing libraries were clustered on 2 lanes of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer's instructions.