title: GSM8018667: FHFR43; Mus musculus; RNA-Seq
library_construction_protocol: P2625 RNA extraction was performed using TRI Reagent® (Molecular Research Center Inc., Cat# TR 188), following the manufacturer's instructions. DNase I treatment was performed to prevent genomic DNA contamination using the TURBO DNA-free™ Kit (ThermoFisher Scientific, Cat# AM1907) manufacturer's instructions. P2625 : Total RNA-Seq libraries were generated from 50 ng of total RNA using Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit and IDT for Illumina RNA UD Indexes, Ligation (Illumina, San Diego, USA), according to manufacturer's instructions. Briefly, abundant ribosomal RNAs were depleted by hybridization to specific DNA probes and enzymatic digestion. The depleted RNAs were purified and fragmented using divalent cations at 94oC for 2 minutes. After random hexamers annealing, fragmented RNAs were then reverse transcribed into first strand complementary DNA (cDNA). Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dTTP in place of dUTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 13 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.