Illumina HiSeq 4000 paired end sequencing; Cooperative blockade of CK2 and ATM kinases drives apoptosis in VHL-deficient renal carcinoma cells / RNA-seq (BRB-seq) of treated 786-O spheroids
Cooperative blockade of CK2 and ATM kinases drives apoptosis in VHL-deficient renal carcinoma cells / RNA-seq (BRB-seq) of treated 786-O spheroids
ccRCC cell lines 786-O was obtained from ATCC. 786-O spheroids were grown for 3 days in RPMI medium containting 10% FCS Three days pre-formed spheroids were treated with DMSO (0.5%) or KU-60019 (10µM) or CX-4945 (7.5 µM) or both during 48h total RNA was extracted using miRVana PARIS kit The 3′ Bulk RNA Barcoding and sequencing (BRB-seq) experiments were performed at the Research Institute for Environmental and Occupational Health (Irset, Rennes, France) according to the published protocol (Alpern et al, Genome Biology, 2019). Briefly, the reverse transcription and the template swiching reactions were performed using 4 µl total RNA at 2.5 ng/µl. RNA were first mixed with 1µl barcoded oligo-dT ( 10µM BU3 primers, Microsynth) , 1μL dNTP (0.2mM) in a PCR plate, incubated at 65 °C for 5 min and then put on ice. The first-strand synthesis reactions were performed in 10 µl total volume with 5 µl of RT Buffer and 0.125 µl of Maxima H minus Reverse Transcriptase (Thermofisher Scientific, #EP0753) and 1 µl of 10μM template switch oligo (TSO, IDT). The plates were then incubated at 42°C for 90 min and then put on ice. After RT, decorated cDNA from multiple samples were then pooled together and purified using the DNA Clean & concentrator-5 Kit (Zymo research, #D4014). After elution with 20 µl of nuclease free water, the samples were incubated with 1 µl Exonuclease I (NEB, #M0293) and 2 µl of 10X reaction buffer at 37°C for 30 min, followed by enzyme inactivation at 80°C for 20 min. Double-strand cDNAs were generated by PCR amplification in 50µl total reaction volume using the Advantage 2 PCR Enzyme System (Clontech, #639206). PCR reaction was performed using 20 µl cDNA from previous step, 5 µl of 10X Advantage 2 PCR buffer, 1 µl of dNTPs 50X, 1 µl of 10 µM LA-oligo (Microsynt), 1 µl of Advantage 2 Polymerase and 22 µl of nuclease-free water following the program (95°C-1 min; 11 cycles: 95°C-15s, 65°C-30s, 68°c-6min; 72°C-10min). Full-length double-stranded cDNA was purified with 30 µl of AMPure XP magnetic beads (Beckman Coulter, #A63881), eluted in 12 µl of nuclease free water and quantify using the dsDNA QuantiFluor Dye System (Promega, #E2670). The sequencing libraries were built by tagmentation using 50 ng of ds cDNA with Illumina Nextera XT Kit (Illumina, #FC-131-1024) following the manufacturer's recommendations. The reaction was incubated 5 min at 55°C , immediately purified with DNA Clean & concentrator-5 Kit (Zymo research) and eluted with 21 µl of nuclease free water. Tagmented library was PCR amplified using 20 µl eluted CDNA, 2.5 µl of i7 Illumina Index, 2.5 µl of 5 µM P5-BRB primer (IDT) using the following program (72°C-3 min; 98°C-30s; 13 cycles: 98°C-10s, 63°C-30s, 72°C-5min). The fragments ranging 300-800 pb were size-selected using SPRIselect (Bekman Coulter, #) (first round 0.65x beads, second 0.56x)with a final elution of 12 µl nuclease-free water.