GSM7991686: UCMSC, rep2; Homo sapiens; RNA-Seq
Total RNAs from EXs and secreting UCMSCs were extracted using Trizol method.  An amount of 50 ng total RNA was used for subsequent library preparation. RNA was used for reversed transcription using random hexamer primers to synthesize the first cDNA strand. The second cDNA strand was synthesized by DNA polymerase using the first cDNA strand as a template. The cDNA fragments were then end-repaired and A-tailed to generate the 5' overhangs for indexing adapter ligation. The A-tailed cDNA fragments were ligated with an indexing adapter, followed by amplification with i5 and i7 indexing primers for sample multiplexing. The indexed libraries were pooled and hybridized with the Illumina exome sequencing panel (Illumina, USA). The library was diluted to 4 nM using the resuspension buffer (RSB), followed by a denaturing step in the presence of NaOH 0.2 N by ration 1:1 (v/v). The denatured library was diluted with denaturing buffer HT1 to achieve a 20 pM denatured library solution. The denatured library was then diluted to the final concentration of 1.3 pM in a total volume of 1.3 mL for sequencing. Sequencing was done on the Nextseq 500/550 system using Nextseq High Output v2.5 (300 cycles) (Illumina, USA).