GSM6909394: Sample 7 - S14, 3h, rep3; Mus musculus; RNA-Seq
Total RNA was extracted using the RNeasy Mini Kit together with the RNase-free DNase Set for DNA digestion (both Qiagen) according to manufacturer instructions Libraries were prepared manually according to the manufacturer's instructions using QuantSeq 3' mRNA-Seq FWD Library Prep Kit. Briefly, 150 ng total RNA was used as input for reverse transcription with oligo(dT) primer containing an Illumina-compatible sequence at its 5' end. After RNA removal, the second strand of the cDNA was synthesized by a random primer containing an Illumina-compatible linker sequence at its 5' end. Finally, individual sample barcodes (dual indexing) for multiplexing were introduced via 15 cycles of PCR (determined by qPCR). All libraries were analyzed for adapter dimers, size distribution and concentration on a Fragment Analyzer (Agilent). After pooling the libraries in an equimolar ratio, the concentration and the size distribution of the lane mix was analyzed by Qubit (Thermo Fisher Scientific) and by Fragment Analyzer (Agilent), respectively. A 2 nM dilution of the lane mix was denatured and diluted for sequencing on a NextSeq 2000 instrument