title: Illumina HiSeq 4000 sequencing; Role of HSP70 in response to (thermo)radiotherapy: analysis of gene expression in canine osteosarcoma cells by RNA-seq
design: Role of HSP70 in response to (thermo)radiotherapy: analysis of gene expression in canine osteosarcoma cells by RNA-seq
library_name: Experiment3_Neg_HT_s
library_construction_protocol: Cells were seeded in a 6-well plate at the density of 50'000 cells/well the day before transfection. Cells were transfected with 25 pmol of custom made siRNA against canine HSP70 (sense 5'-CCACCUACUCGGACAACCATT-3', antisense 5'-UGGUUGUCCGAGUAGGUGGTG-3'; Thermo Fisher Scientific) and with Silencer Select Negative Control No. 1 siRNA (Cat. No. 4390843, Thermo Fisher Scientific) using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific). 24 hours after transfection medium was exchanged and cells were treated with hyperthermia, irradiation or combination of both as described above. RNA and protein lysates samples were collected 24 hours after treatment with irradiation. RNA was isolated using RNeasy Mini Kit (Qiagen). RNA concentration was measured using NanoDrop and adjusted to 100 ng/ml, samples were stored at -80C until analysis.  The RNA sequencing  The quality of the RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Total RNA samples (600 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR.  The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA).  The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.
attributes.tag: Experimental Factor: RNA interference
attributes.value: negative siRNA
attributes.tag: Experimental Factor: stimulus
attributes.value: hyperthermia