Qiashredder kit

Cells were washed twice with PBS and homogenized with QIAshredder Kit (Qiagen). Total RNA was extracted with RNeasy Plus Mini Kit (Qiagen) which include a genomic DNA elimination step. cDNA synthesis was performed with iScript cDNA syntesys kit (Biorad). Quantitative real-time PCR (qPCR) was performed in triplicate using iQ SYBR Green Supermix kit and CFX96^™ Real-Time System (Biorad). Thermocycling conditions were: 95 °C for 3 min, followed by 45 cycles at 95 °C for 10 s, 60 °C for 30 s. Gene expression was normalized (GAPDH or VE-Cad) and relative expression was calculated using the ΔΔCt method. A complete primer list is reported in Supplementary Table 1.

Cell pellets (1.6 × 10⁶ cells) were first lysed and homogenized using the QIAshredder kit (Qiagen, Courtaboeuf, France), then total RNAs were extracted using the RNeasy mini kit (Qiagen) according to manufacturer’s instructions. The total RNA was quantified using a Nanodrop^®ND-1000 spectrophotometer (Thermo Fisher Scientific, Illkirch, France). The quality and integrity of RNA samples were assessed using the 2100 Bioanalyzer and RNA 6000 Nano LabChip kit series II (Agilent Technologies, Les Ulis, France). The RNAs extracted were of good quality, and the RNA integrity number (RIN) was >9 in all cases.

Cells were lysed over ice in RLT buffer from RNeasy Mini Kit (QIAGEN, #74104) supplemented with 10 µL/mL β-mercaptoethanol. Lysates were homogenized using the QIAshredder kit (QIAGEN, #79656) and stored at -80°C until extraction. All RNA extractions were done using the RNeasy Mini Kit (QIAGEN, #74104) including the optional DNase treatment step (RNAse-Free DNase Set, QIAGEN, #79254) and eluted in 35-50 µL RNAse-free water. RNA concentration was quantified with the Nanodrop 2000c. cDNA was made using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, #10704217). For each experiment, equal quantity of RNA was added across samples, using up to 9µL RNA. qPCR reactions comprised of 1 volume sample cDNA to 3 volumes master mix (Power SYBR™Green PCR Master Mix (Applied Biosystems, #4367659), forward and reverse primer mix, and nuclease-free water at a ratio of 5 µL: 1 µL: 2 µL respectively). Primers used are listed in supplementary table 5. Reactions were in run in triplicate in 384-well format, 6-8 µL/sample in the Applied Bioscience QuantStudio™ 5. Default cycling conditions were used (supplementary table 6). Analysis of gene expression was performed by normalization of Ct values to the average Ct value of two housekeeper genes, VIPAR and UBE4A, and displayed as fold difference as calculated by 2^-ΔCt.

RNA was extracted using the Qiagen Qiashredder kit (79654) and the Qiagen RNeasy Mini Kit (74104) as per the manufacturer’s instructions (Qiagen Inc., Valencia, CA, USA). The RNA was DNase treated following the protocol in the RNeasy Mini Kit with Qiagen RNase-free DNase I (79254). Indexed PolyA libraries were prepared using 1 μg of total RNA and 13 cycles of amplification in the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat No: E7420S). RNA Integrity Values scores were >9 for all samples. Libraries were quantified by qPCR using a Kapa Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems, Wilmington, MA, USA, Cat No: KK4835). Pooled libraries were clustered at 15 pmol/l on the cBot and 2×100 bp sequencing was carried out using the High Throughput mode of a HiSeq 2500 using TruSeq SBS Kit v3 chemistry (Illumina)