title: GSM5542616: CD19 minigene library RNA-seq 02; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were harvested after 24hrs of the electroporation and RNA was isolated via the RNeasy Mini Kit (Qiagen). 20 µg isolated RNA was poly-A-selected using Dynabeads Oligo (dT)25 beads (Invitrogen) according to manufacturer's recommendations. Reverse transcription was performed on 500 ng poly-A-selected RNA with RevertAid Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer's recommendations. To prevent chimeric amplicons, the RNA-seq libraries were amplified via emulsion PCR (Williams, 2006) using the Phusion DNA Polymerase (NEB). The following primers containing Illumina adapters were used in the PCR: 5'- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGGAACCTCTAGTGGTGAAGG-3' (fwd) 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCCGCCAGTGTGATGGATATC-3' (rev) under following conditions: : 98 °C for 30 s, 25 cycles of [98 °C for 10 s, 63 °C for 20 s, 72 °C for 1 min] and final extension at 72 °C for 5 min. Amplicons were purified using Agencourt AMPure XP beads (Backman Coulter). Purified products were analysed on the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and quantified using the Qubit assay (Thermo Fisher Scientific).