GSM2716990: Ingolf40: undiff_KO_female rep2; Mus musculus; RNA-Seq
Library was constructed as described (Vallaster et al., 2016) RNA libraries were prepared for sequencing using standard Illumina protocols.  A total of 187 samples were distributed on two 96-well plates (plus 5 mock wells) and thawed at RT for 10 minutes prior to RNA purification using Ampure RNA beads (Beckmann-Coulter). RNA samples were re-suspended in solution containing 3’ RT primer and dNTPs. Reverse transcription was performed with SSII (Life Technologies), whose terminal transferase activity allows incorporation of a PCR binding site at the 3’ end of the cDNA using a template-switching oligonucleotide (custom synthesized from Exiqon) as a template. Subsequently, cDNA was amplified using 12 cycles of PCR, followed by tagmentation with Nextera kit (Illumina). Final libraries were amplified by 12 cycles of PCR and sequenced on a NextSeq 500.