title: GSM4007362: ciprofloxacin-hi rep5; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was prepared using PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. DNA removal was done on column with PureLink DNase (Invitrogen). cDNA synthesis using High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA) according to product insert. qPCR was completed using TaqMan® assays with Fast Advanced Master Mix (Applied Biosystems) as per manufacturer's guidelines.  RNA samples were quantified and quality assessed using an Advanced Analytical Fragment Analyzer. 20ng of totalRNA was used for library preparation with ERCC Spike-in control Mix A (Ambion 10-6 final dilution). All steps were performed on a Tecan EVO150. 3'DGE-custom primers 3V6NEXT-bmc#1-24 are added to a final concentration of 1.2uM. (5'-/5Biosg/ACACTCTTTCCCTACACGACGCTCTTCCGATCT[BC6]N10T30VN-3' where 5Biosg = 5' biotin, [BC6] = 6bp barcode specific to each sample/well, N10 = Unique Molecular Identifiers, Integrated DNA technologies). After addition of the oligonucleotides, samples were denatured at 72C for 2 minutes followed by addition of SMARTScribe RT per manufacturer's recommendations with Template-Switching oligo5V6NEXT (12uM, [5V6NEXT : 5'-iCiGiCACACTCTTTCCCTACACGACGCrGrGrG-3' where iC: iso-dC, iG: iso-dG, rG: RNA G ]) and incubation at 42C for 90' followed by inactivation at 72C for 10'. Following the template switching reaction, cDNA from 24 wells containing unique well identifiers were pooled together and cleaned using RNA Ampure beads at 1.0X. cDNA was eluted with 90 ul of water followed by digestion with Exonuclease I at 37C for 45 minutes, inactivation at 80C for 20 minutes. Single stranded cDNA was then cleaned using RNA Ampure beads at 1.0X and eluted in 50ul of water. Second strand synthesis and PCR amplification was done using the Advantage 2 Polymerase Mix (Clontech) and the SINGV6 primer (10 pmol, Integrated DNA Technologies 5'-/5Biosg/ACACTCTTTCCCTACACGACGC-3' ). 12 cycles of PCR was performed followed by clean up using regular SPRI beads at 1.0X, and was eluted with 20ul of EB. Successful amplification of cDNA was confirmed using the Fragment Analyzer. Illumina libraries are then produced using standard Nextera tagmentation substituting P5NEXTPT5-bmc primer (25μM, Integrated DNA Technologies, (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T*-3' where * = phosphorothioate bonds. ) in place of the normal N500 primer. Final libraries were cleaned using SPRI beads at 1X and quantified using the Fragment Analyzer and qPCR before being loaded for paired-end sequencing using the Illumina NextSeq500.