title: NextSeq 550 sequencing; RNA-seq of human patient-derived xenograft pancreatic cancer PancA6L cells cultured for 14 days with Galactose versus PancA6L cells cultured for 14 days with Glucose
design: RNA-seq of human patient-derived xenograft pancreatic cancer PancA6L cells cultured for 14 days with Galactose versus PancA6L cells cultured for 14 days with Glucose
library_construction_protocol: PancA6L cells cultured for 14 days with Glucose (0.9 g/L) or Galactose (0.9 g/L) were washed with 1X PBS twice and then resuspended in 500µl 4M guanidine thiocyanate (GTC) supplemented with 8µl/ml β-mercaptoethanol and 2µl/ml ultrapure glycogen (Thermo, Cat no. 10814010 [20 µg/µl]). This protocol was repeated on three different occasions to obtain biological triplicates.  PDX PanA6L cells were grown as adherent 2D monolayers with RPMI medium supplemented with 10% fetal bovine serum (FBS) and 50 units/mL of penicillin and streptomycin in a 5% CO2 incubator at 37 degrees C.   PDX PancA6L monolayers were cultured with either 1) glucose-free DMEM medium (Dulbecco´s Modified Eagle Medium, Thermo Fisher Scientific) supplemented with 5mM glucose (0.9 g/L), 10% FBS, 50 units/mL of penicillin and streptomycin and 1mM of pyruvate [Glucose: OXPHOS-independent conditions] or 2) glucose-free DMEM medium (Thermo Fisher Scientific) supplemented with 5mM galactose (0.9 g/L), 10% FBS, 50 units/mL of penicillin and streptomycin and 1mM of pyruvate [Galactose: OXPHOS-competent enriched conditions]. Sugar concentrations of 5mM were chosen to mimic physiological sugar levels (glucose, 5mM) and to avoid potential biological artifacts mediated by supraphysiological sugar levels. Media for both conditions were changed every day, over a period 14d, after which, cells were collected for further processing and analysis.  Total RNA was purified suing the GTC standard protocol extraction method with acidic phenol and chloroform. RNA was precipitated at -80C with Isoproponal containing 3µl of linearized polyacrylamide. RNA pellets were washed sequentially with 80% and 100% ETOH, dried RNA pellets were re suspended in nuclease free H2O and RNA integrity analyzed via Bionanalizer.  Sequencing libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) as recommended by the kit manufacturer. Briefly, polyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters. Adapter-ligated library was completed by PCR with Illumina PE primers. {This kit generates directional libraries stranded in the antisense orientation [the read1 (the only read in single read format) has the antisense orientation (--library-type fr-firststrand in TopHat, --stranded=reverse in HTSeq]} The resulting purified cDNA libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina instrument by following manufacturer's protocols.