GSM5708726: CIC-KO1 (IDH1-WT) RNA-seq rep 3; Homo sapiens; RNA-Seq
To remove cytoplasmic and mitochondrial ribosomal RNA (rRNA) species from total RNA, the NEBNext rRNA Depletion Kit for Human/Mouse/Rat was used (NEB, E6310X). Enzymatic reactions were set-up in a 96-well plate (Thermo Fisher Scientific) on a Microlab NIMBUS liquid handler (Hamilton Robotics, Reno, Nevada, USA). 100ng of DNase I-treated total RNA in 6 µl was hybridized to rRNA probes in a 7.5 µl reaction. Heat-sealed plates were incubated at 95 °C for 2 min followed by incremental reduction in temperature by 0.1 °C per s to 22 °C (730 cycles). The 15 rRNA in DNA hybrids were digested using RNase H in a 10 µl reaction incubated in a thermocycler at 37 °C for 30 min. To remove excess rRNA probes (DNA) and residual genomic DNA contamination, DNase I was added in a total reaction volume of 25 µl and incubated at 37 °C for 30 min. RNA was purified using RNA MagClean DX beads (Aline Biosciences, Woburn, Massachusetts, USA) with 15 min of binding time, 7 min clearing on a magnet followed by two 70% ethanol washes, 5 minutes to air dry the RNA pellet and elution in 36 µl DEPC water. The plate containing RNA was stored at -80 °C prior to cDNA synthesis. First-strand cDNA was synthesized from the purified RNA (minus rRNA) using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher, USA) and random hexamer primers at a concentration of 8 ng/µl along with a final concentration of 0.04 µg/µl Actinomycin D, followed by PCR Clean DX bead purification on a Microlab NIMBUS robot (Hamilton Robotics, USA). The second strand cDNA was synthesized following the NEBNext Ultra Directional Second Strand cDNA Synthesis protocol (NEB) that incorporates dUTP in the dNTP mix, allowing the second strand to be digested using USERTM enzyme (NEB) in the post-adapter ligation reaction and thus achieving strand specificity. cDNA was fragmented using Covaris LE220 sonication for 100 s (2 x 50 s) at a “Duty cycle” of 30%, 450 Peak Incident Power (W) and 200 Cycles per Burst in a microTUBE Strip (P/N: 520053) to achieve 200–250 bp average fragment lengths. The paired-end sequencing library was prepared following Canada's Michael Smith Genome Sciences Centre's strand-specific, plate-based library construction protocol on a Microlab NIMBUS robot (Hamilton Robotics, USA). In brief, the sheared cDNA was subject to end-repair and phosphorylation in a single reaction using an enzyme premix (NEB) containing T4 DNA polymerase, Klenow DNA Polymerase and T4 polynucleotide kinase, and incubated at 20 °C for 30 min. Repaired cDNA was purified in 96-well format using PCR Clean DX beads (Aline Biosciences, USA), and 3'-A-tailed (adenylated) using Klenow fragment (3' to 5' exo minus) and incubation at 37 °C for 30 min prior to enzyme heat inactivation. Illumina PE adapters were ligated at 20 °C for 15 min. The adapter-ligated products were purified using PCR Clean DX beads, then digested with USERTM enzyme (1 U/µl, NEB) at 37 °C for 15 min followed immediately by 13 cycles of indexed PCR using Phusion DNA Polymerase (Thermo Fisher Scientific Inc. USA) and Illumina's PE primer set. PCR parameters were: 98 °C for 1 min followed by 13 cycles of 15 s at 98 °C, 30 s at 65 °C and 30 s at 65 °C, and then 72 °C for 5 min. The PCR products were twice purified and size-selected using a 1:1 PCR Clean DX beads-to- 16 sample ratio, and the eluted DNA quality was assessed using an Agilent DNA1000 Assay (Agilent, USA) and quantified using a Quant-iT dsDNA High Sensitivity Assay Kit on a Qubit fluorometer (Invitrogen). Libraries (3 replicates per cell line) were then pooled and size- corrected using a final molar concentration calculation for Illumina HiSeq2500 sequencing with paired-end 75 base reads.