description: Protocols: Outer root sheath keratinocyte (ORS-KCs) were isolated from plucked hair follicles (HFs) using enzymatic digestion as previously described (PMID: 21593771) with minor alterations. Briefly human HFs were plucked from the temporal scalp, washed in PBS with Gentamycin and incubated for up to 45 min with 0.25% Trypsin EDTA. Following removal of the ORS, cells were seeded onto a feeder layer of human dermal fibroblasts pre-treated with 4µg/ml Mitomycin C (Sigma) and grown in ORS media comprising of a 3:1 mix of DMEM Glutamax (Gibco) and Ham's F-12 Nutrient mix (Gibco) supplemented with 10% foetal calf serum (Gibco), 5µg/ml insulin, 0.4µg/ml hydrocortisone, 2.4 µg/ml adenine, 2nM T3, 0.1nM cholera toxin, 10ng/ml EGF, 1mM ascorbyl-2-phosphate, Penicillin and 25µg/ml gentamycin. Approximately 1-2 weeks later when cells had established colonies, they were removed from feeder layers and subsequently cultured in epilife media (Gibco) and used between passages 2-5. This study of outer root sheath keratinocyte (ORS-KCs) had 4 conditions with n=3. Conditions: Control, T0901317, doxorubicin (DOX), T0901317+DOX. ORS-KCs were isolated from plucked hair follicles (HFs) using enzymatic digestion as previously described (PMID: 21593771) with minor alterations. Briefly human HFs were plucked from the temporal scalp, washed in PBS with Gentamycin and incubated for up to 45 min with 0.25% Trypsin EDTA. Following removal of the ORS, cells were seeded onto a feeder layer of human dermal fibroblasts pre-treated with 4µg/ml Mitomycin C (Sigma) and grown in ORS media comprising of a 3:1 mix of DMEM Glutamax (Gibco) and Ham's F-12 Nutrient mix (Gibco) supplemented with 10% foetal calf serum (Gibco), 5µg/ml insulin, 0.4µg/ml hydrocortisone, 2.4 µg/ml adenine, 2nM T3, 0.1nM cholera toxin, 10ng/ml EGF, 1mM ascorbyl-2-phosphate, Penicillin and 25µg/ml gentamycin. Approximately 1-2 weeks later when cells had established colonies, they were removed from feeder layers and subsequently cultured in epilife media (Gibco) and used between passages 2-5. ORS-KCs were cultured for 72 hours in the presence or absence of 5 µM T0901317 before the addition of 5 µM DOX for 4 hours to detect early changes in response to DOX. Total RNA was extracted using RNeasy® Microkit (Qiagen, UK) according to manufacturer's protocol. Truseq stranded mRNA Assay. Paired end.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-02-19T04:06:46Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-07-16T10:03:21Z
attributes.tag: External Id
attributes.value: SAMEA7064192
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF MANCHESTER
attributes.tag: INSDC first public
attributes.value: 2021-02-19T04:06:46Z
attributes.tag: INSDC last update
attributes.value: 2020-07-16T10:03:21Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9362:Sample 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: keratinocyte
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: 2
attributes.tag: organism part
attributes.value: skin of scalp
attributes.tag: sample name
attributes.value: E-MTAB-9362:Sample 8
attributes.tag: sampling site
attributes.value: outer root sheath
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male