Samples C1,2,3-HeLa and C1,2,3,4,5-HF73 were prepared from cells incubated at 37ºC for 3.5h with 0.25% DMSO. Samples H1,2,3-HeLa and H1,2,3,4,5-HF73 were prepared from cells incubated at 37ºC for 2 h with 0.25% DMSO, then heat treated at 43ºC for 30min in a water bath and further incubated at 37ºC for 1h. Samples HT1,2,3-HeLa and HT1,2,3,4,5-HF73 were prepared from cells incubated at 37ºC for 2 h with 25 mM IHSF115, then heat treated at 43ºC for 30min in a waterbath and further incubated at 37ºC for 1h.
Growth protocol
Cells were maintained in a humidified 5% CO2 atmosphere at 37ºC. Hela cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Basel, Switzerland) supplemented with 10%(v/v) fetal bovine serum, 10U/ml penicillin and 0.01mg/ml streptomycin. HSF2-deficient cell line HF73 was prepared by transfection of HeLa cells with equal amounts of the three plasmids of the human HSF2 sgRNA CRISPR/Cas9 All-in-One Lentivector set of Applied Biological Materials.Transfections employed Lipofectamine reagent (Invitrogen). Cell line HF73 was isolated after selection with 1.3mg/ml puromycin. HF73 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%(v/v) fetal bovine serum, 10U/ml penicillin, 0.01mg/ml streptomycin and 1.3mg/ml puromycin.
Extracted molecule
total RNA
Extraction protocol
Total RNA prepared using the RNeasy Mini Kit (Qiagen, Hilden, Germany)
Label
biotin
Label protocol
Total RNA was processed using the GeneChip WT PLUS Reagent kit (Applied Biosystems, Waltham, MA).
Hybridization protocol
Biotinylated cRNA was hybridized with GeneChip Human Gene 2.0 ST Array (Applied Biosystems), following the manufacturer´s instructions.
Scan protocol
Arrays were scanned with a GeneChip scanner 3000 7G (Applied Biosystems).
Data processing
Raw data were normalized and gene levels analyzed using the RMA algorithm (Transcriptome Analysis Console Software; Applied Biosystems).
