ERC000011
ENA-CHECKLIST
2023-11-28T17:00:04Z
ENA-FIRST-PUBLIC
2023-11-28T17:00:04Z
ENA-LAST-UPDATE
SAMEA113907501
External Id
UOM
INSDC center name
2023-11-28T17:00:04Z
INSDC first public
2023-11-28T17:00:04Z
INSDC last update
public
INSDC status
ERS15901668
SRA accession
E-MTAB-13106:CP3
Submitter Id
ArrayExpress
broker name
natural killer cell
cell_type
not collected
collection date
human
common name
Protocols: Peripheral blood leukocyte cones from healthy human donors were acquired from the National blood service under ethics license REC 05/Q0401/108 (University of Manchester). Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation using Ficoll-Paque (GE Healthcare; Life Sciences) and NK cells isolated by negative selection (NK cell isolation kit; Miltenyi Biotech). NK cells were cultured at 37⁰C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) medium (Sigma) containing 30% Ham’s F-12, 10% human serum (Sigma), 1% nonessential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine, 50 U/mL penicillin-streptomycin, and 50 μM 2-mercaptoethanol (Gibco) (NK cell clone medium). NK cells were also supplemented with 200 U/mL interleukin-2 (Roche) and rested for 6 days prior to experiments. To investigate the effects of PGE₂, NK cell cultures were additionally supplemented with 500 ng/mL synthetic PGE₂ (Sigma) 24 hours prior to cell harvesting and lysis. NK cells were cultured at 37⁰C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) medium (Sigma) containing 30% Ham’s F-12, 10% human serum (Sigma), 1% nonessential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine, 50 U/mL penicillin-streptomycin, and 50 μM 2-mercaptoethanol (Gibco) (NK cell clone medium). NK cells were also supplemented with 200 U/mL interleukin-2 (Roche) and rested for 6 days prior to experiments. After being rested for six days the NK cells were left untreated or supplemented with 500 ng/mL synthetic PGE₂ (Sigma) 24 hours prior to cell harvesting and lysis. NK cells were re-suspended in RLT buffer (RNeasy lysis buffer; Qiagen) containing 0.01% β-mercaptoethanol to lyse cells and stored at -80⁰C prior to RNA extraction. RNA was extracted from thawed samples using the RNeasy mini kit (Qiagen) as per the manufacturer’s instructions. mRNA libraries were prepared using the Illumina stranded mRNA prep kit.
description
adult
developmental stage
normal
disease
not collected
geographic location (country and/or sea)
2
individual
Homo sapiens
organism
Homo sapiens
organism
blood
organism part
Homo sapiens
scientific_name
CP3
title