2025-12-31
ENA first public
ERC000011
ENA-CHECKLIST
ACTREC - Tata Memorial Centre
INSDC center name
public
INSDC status
ERS18104726
SRA accession
E-MTAB-13769:RNA-seq of HL-60/MX2 cell line: siControl3
Submitter Id
ArrayExpress
broker name
HL60/MX2
cell_line
Peripheral blood leukocyte
cell_type
not collected
collection date
human
common name
Protocols: PRKDC knockdown: 0.2X106 cells/mL were seeded per well in a 6-well plate and transfected the next day with siRNA at 80pmols PRKDC (siRNA select-ThermoFisher, 4390824) per well using the standard 6-well protocol for Lipofectamine3000 reagent (Invitrogen, L3000015). Cells were collected 48 h post-transfection and tested for the knockdown of the respective proteins by qPCR and immunoblotting. RNA was isolated from the cell pellet resuspended in TRIzol Reagent (Thermo Fisher Scientific) using the chloroform-isopropanol method. The RNA samples were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high-sensitivity RNA screen tape to determine the RNA integrity number (RIN). Samples with 28S/18S peak integrated area ratios >2 were used for library preparation. 1.5 μg of QC verified RNA samples was used for poly (A) mRNA isolation from total RNA using NEB Next Poly (A) mRNA Kit (Cat. no. NEB# 7490) according to the manufacturer’s protocol. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760L) was used to construct double-stranded cDNA libraries from the polyA RNA fraction, using RNA fragmentation, first strand cDNA synthesis with random primers, second strand cDNA synthesis, end repair of double-stranded cDNA, adapter ligation, removal of excess adapter using sample purification beads, PCR enrichment of adapter ligated DNA, and clean-up of PCR products as instructed by the manufacturer. The cleaned libraries were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high sensitivity D1000 screen tape to determine the size range of the fragments and the average library size.
description
adult
developmental stage
adult acute myeloid leukemia
disease
resistant
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
blood
organism part
Homo sapiens
scientific_name
RNA-seq of HL-60/MX2 cell line: siControl3
title