GSM2532927: PTL_4_Pax_mRNA; Homo sapiens; RNA-Seq
Whole blood RNA was isolated from PAXgene tubes using microRNA isolation kit (Qiagen) using TRIsol LS (Invitrogen) following manufacturer’s instructions. RNA library preparation was performed following the NEB NEBNext Ultra Directional Library Preparation protocol.400 ng of total RNA was used as the input material and enriched for poly-A mRNA, fragmented into the 200-300-bases range for 4 minutes at 94oC and converted to double stranded cDNA, end-repaired and adenylated at the 3’ to create an overhang A to allow for ligation of Illumina adapters with an overhang T; library fragments were amplified under the following conditions: initial denaturation at 98oC for 10 seconds, followed by 15 cycles of 98oC for 10 seconds, 60oC for 30 seconds and 72oC for 30 seconds, and finally an extension step for 5 minutes at 72oC; at the amplification step, each sample were amplified with a different barcoded adapters to allow for multiplex sequencing. One ul of the final RNA libraries was loaded on a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) to check for size; RNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). Libraries were pooled in equimolar quantities and paired-end sequenced on a High Throughput Run Mode flowcell with the V4 sequencing chemistry on an Illumina HiSeq 2500 platform following Illumina’s recommended protocol to generate paired-end reads of 126-bases in length.