title: GSM5471938: TeloHAEC CTRL rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were harvested 48 h post-transfection and total RNA was extracted using the RNeasy Midi Kit (Qiagen). PolyA RNA was purified from total RNA using Dynabeads Oligo(dT)25 beads (Invitrogen) with 2:1 bead to total RNA volume ratio. The isolated polyA RNA was treated with Turbo DNaseI (Ambion) and purified using RNeasy MinElute clean up kit (Qiagen). Reverse transcription of reporter transcript mRNA was performed using a gene-specific primer containing a 10 nt unique molecular identified (UMI). After reverse transcription, RNA was remove by RNase A treatment and the cDNA was purified with AMPure XP beads using a 1.8:1 beads to cDNA ratio. The cDNA-s were amplified using a reporter-specific PCR amplicon targeting an exon-exon junction to minimize potential contribution from plasmid DNA. The junction PCR products were purified using AMPure XPbeads with beads to sample ratio of 0.8. A second PCR step was run to add the Illumina multiplexing indices for sequencing (NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 and 2). The final library PCR product was purified using SPRIselect beads (Beckman) with a bead to sample ratio of 0.8. Sequencing was performed on an Illumina NextSeq 500 platform as a paired-end dual-index run using the following read length setup: 37bp (insert sequence), 10bp (the UMI in place of the i7 index), 8bp (i5 index for Illumina demultiplexing), 37bp (insert sequence).