Total RNA was extracted using TRIzol® Reagent following the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The RNA purity and quantity was measured with a NanoDrop 2000C® spectrophotometer (Thermo Fischer Scientific AG, Reinach, CH). Further purification of RNA was performed by the RNeasy® Mini Kit (Qiagen GmbH, Hilden, DE). RNA integrity was assessed with the Agilent 2200 TapeStation System. The RNA integrity numbers (RIN) ranged from 7.2 to 9.5. The quality and quantity of isolated RNA was determined with Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000ng) were poly-A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of enriched libraries were validated using the Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq PE Cluster Kit v4-cBot-HS or TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc., California, USA) were used for cluster generation using 10 pM of pooled normalized libraries on the cBOT System. Sequencing was performed on the Illumina HiSeq 2500 single end 125 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc.).
GEO Accession
GSM2608007