GSM8750883: SC, 34WT, differentiation, biol rep 3; Sus scrofa; RNA-Seq
Total RNA was extracted from cells in PROL and DIFF conditions using the NEB Monarch RNA Miniprep Kit, the protocol was performed accordingly to the instructions provided by the manufacturer. 25 ng of total RNA was used. polyA+ RNA s were selected during cDNA synthesis through annealing to a polydT oligo containing an unique molecular idenitifer (UMI) and a well-barcode. Second strand synthesis was done by RT-PCR using a template switch oligo with an integrated Illumina adapter sequence. After purification and exonuclease°I treatment, the library pool was amplified by PCR and purified again. To achieve optimal fragment lengths, NexteraXT DNA library prep Kit (Illumina) was used. With the Nextera transposon DNA was tagmented, a process that fragments and tags DNA with adapter sequences. For amplification of the library, a 3' enrichment PCR was performed during which i7 and i5 adapters were attached. After purification, the quantity of the library pool (Qubit Fluorometer 4.0, Thermo Fisher) and average fragment size (LabchipGX Touch 24, Perkin Elmer/Revvity) were measured. The resulting library was sequenced on a NextSeq550Dx using a 75-cycle high output kit (Read 1:16°cycle – UMI + barcode; Read 2: 76°cycles – Insert).