title: CP6
organism: Homo sapiens
description: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-11-28T17:00:04Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-11-28T17:00:04Z
attributes.tag: External Id
attributes.value: SAMEA113907499
attributes.tag: INSDC center name
attributes.value: UOM
attributes.tag: INSDC first public
attributes.value: 2023-11-28T17:00:04Z
attributes.tag: INSDC last update
attributes.value: 2023-11-28T17:00:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-13106:CP6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: natural killer cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: individual
attributes.value: 3
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-13106:CP6
attributes.tag: scientific_name
attributes.value: Homo sapiens