title: Illumina HiSeq 4000 paired end sequencing; LXR mediated-signalling protects human hair follicles against doxorubicin-induced toxicity ex vivo
design: LXR mediated-signalling protects human hair follicles against doxorubicin-induced toxicity ex vivo
library_construction_protocol: 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.