biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsYongxiang FangIllumina HiSeq 2000RNA-seq of coding RNAhumanhumanhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-4104The KBM-7 reference clone B (product no. P00174E07) and the KBM-7-NuKO derivative (P01289H04) in which the NUDT2 gene has been inactivated by retroviral gene-trap insertion were obtained from Haplogen and maintained. Three independent samples of total RNA were prepared from both KBM-7 and NuKO cells and sequenced using Illumina HiSeq2000.biostudies-arrayexpressSequencing - The template DNA was denatured according to the protocol described in the Illumina cBot User guide and loaded at 9 pM concentration. To improve sequencing quality control 1% PhiX was spiked-in. The sequencing was carried out on one lane of an Illumina HiSeq 2000 with version 3 chemistry generating 2 100 bp paired end reads.Library Construction - The depleted RNA was used as input material for the ScriptSeq v2 RNA-Seq Library Preparation protocol. Following 15 cycles of amplification the libraries were purified using Ampure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyzer.Nucleic Acid Extraction - 10ug of total RNA was DNase treated using Ambion Turbo DNA-free Kit, and subsequently purified using Ampure XP beads. 2ug of the DNase treated total RNA was then used as input material into RNA depletion using the Ribozero Complete Gold (human/mouse/rat) and purified again with Ampure XP beads. Successful depletion was then QCd using Qubit and Agilent 2100 Bioanalyzer.Growth Protocol - Cells (2 E+5 ) were seeded into 25 square cm flasks containing 7 mL of growth medi um . Chemical agents were added as stated and cells grown for 96 h at 37 o C after which cultures were centrifuged at 500 g for 5 min, cells resuspended in fresh medium, and counted using a haemocytometer. Average counts were normalized to the cell coun t of the untreated culture.Sample Collection - The KBM-7 reference clone B (product no. P00174E07) and the KBM-7- NuKO derivative (P01289H04) in which the NUDT2 gene has been inactivated by retroviral gene-trap insertion were obtained from Haplogen and maintained at 37 o C in 5% (v/v) CO 2 /air in Isocoves modified Eagle medium (IMEM, Sigma) supplemented with 10% (v/v) Foetal Bovine Serum (Sigma), 2 mM L-glutamine (Sigma) and 100 g mL -1 penicillin-streptomycin (Sigma)OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesYongxiang FangfalseDfifferential gene expression in human KBM-7 clone B and KBM-7-NuKO (NUDT2 KO) cellsThe KBM-7 reference clone B (product no. P00174E07) and the KBM-7-NuKO derivative (P01289H04) in which the NUDT2 gene has been inactivated by retroviral gene-trap insertion were obtained from Haplogen and maintained. Three independent samples of total RNA were prepared from both KBM-7 and NuKO cells and sequenced using Illumina HiSeq2000.2015-12-09T00:00:00Z2022-03-04T16:34:26.363Z2022-03-04T16:34:26.363ZE-MTAB-4104ERP013422EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003738EFO_0004184