GSM6065821: PS+ day10 biol repl2; Mus musculus; RNA-Seq
Cells were lysed in Trizol. RNA purification was done using Monarch Cleanup Kits (New England Biolabs). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, first strand cDNA was synthesized using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. After fragmentation, the first-strand cDNA was purified and the Illumina TruSeq sequencing adapter was ligated in a strand specific manner to the 5' ends of the cDNA fragments. The cDNA was PCR amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (cycle numbers are indicated in Table 2). The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool in the size range of 300 – 400 bp was eluted from a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pools were sequenced in four runs on an Illumina NextSeq 500 system using 1x75 bp read length.