title: GSM9328146: SLAM-seq MYC-mAID auxinole (ctrl) rep 3; Homo sapiens; OTHER
library_construction_protocol: Cells were washed twice with fresh medium and scraped stepwise in 2 × 0.5 ml of ice cold TRIzol LS Reagent (Invitrogen, 10296010) and proceeded immediately to the next step. For RNA isolation, we followed the manufacturer's instructions of SLAM-seq Kinetics Kit-Anabolic Kinetics Module (Lexogen, 061.24) except for the following modifications: Samples were incubated for 5 min at 65°C and mixed with 200 μl of chloroform:isoamyl alcohol mix (24:1) (Sigma-Aldrich, 25666), before further incubation for 15 min at 65°C under strong agitation (2000 rpm) and repeated vortexing. RNA in the aqueous phase was separated by centrifugation at 16000g for 15min at 4°C, supplemented with reducing agent and again extracted with one amount of chloroform:isoamyl alcohol mix (24:1) before precipitation overnight at −80°C (in the presence of reducing agent). RNA was pelleted for 20 minutes at 16000g washed twice with 1 and 0.18 ml of 80% EtOH (plus reducing agent), respectively, and pelleted for 10 min at 7500g. Resuspension in the elution buffer (provided by the SLAM-seq Kinetics Kit) was facilitated by incubation for 15 min at 55°C, 10 min on ice and gentle pipetting to avoid fragmentation of RNA. RNA was quantified with the Qubit RNA HS Assay Kit (Invitrogen, Q32852) and its integrity was controlled using the RNA 6000 nano kit (Agilent, 5067-1511) on a Bioanalyzer 2100 (Agilent) . To facilitate normalization of the sequencing reads, the isolated RNA was mixed with ERCC RNA Spike In-Mix (Invitrogen, 4456740). Specifically, RNA (4.5 μg) was mixed with 2 μl of 1:10 diluted ERCC RNA Spike In-Mix and the elution buffer was added to fill up to 10 μl. Next 4.5 μg RNA mixed with ERCC was taken, the elution buffer added up to 15 μl and alkylation was performed by addition of iodoacetamide following the manufacturer's instructions. Once again Qubit RNA HS Assay Kit (Invitrogen, Q32852) was used to quantify the RNA, and RNA integrity was controlled using the RNA 6000 nano kit (Agilent, 5067-1511) on a Bioanalyzer 2100 (Agilent). Ribosomal RNAs (rRNAs) were depleted from 950 ng of alkylated RNA using the RiboCop (HMR) kit (as part of the CORALL Total RNA Seq V2 12nt B1 UDI12B_0001-0024, Lexogen, 184.24) following the manufacturer's instructions, except for increasing the volume for RNA elution to 16 μl. Removal of rRNAs was confirmed using the RNA 6000 Pico Kit (Agilent, 5067-1513) on a Bioanalyzer 2100 (Agilent). To prepare cDNA libraries spanning the whole transcript length, the CORALL Total RNA Seq V2 12nt B1 UDI12B_0001-0024 (Lexogen, 184.24) was used, which includes reverse transcription with displacement stop primers. Ten microliters of the rRNA-depleted RNA was used as input material, and the final library was amplified by 15 PCR cycles as defined from the PCR Add-on and reamplification Kit V2 for Illumina (Lexogen, 208.96). DNA concentration and molarity were determined by Qubit dsDNA HS Assay Kit (Invitrogen, Q33231) and Bioanalyzer High Sensitivity DNA Kit (5067-4626) on a Bioanalyzer 2100 (Agilent), respectively. Libraries were pooled and sequenced using the NextSeq™ 1000/2000 P2 XLEAP-SBS™ Reagent Kit (100 Cycles) (Illumina, 20100987). The sequencing run was paired-end and dual index with 2 x 53-bp reads.