title: GSM6659832: p0 PP CINI 3; Homo sapiens; RNA-Seq
library_construction_protocol: Qiagen RNeasy Total RNA with an integrity number of ≥ 8.8 was used. mRNA was isolated from 1 ug total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (NEBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB). For ligation custom adaptors were used 1: (Adaptor-Oligo 5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3', Adaptor-Oligo 2: 5'-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3'). After ligaton adapters were depleted by an XP bead purification (Beckman Coulter) adding bead in a ratio of 1:1. Indexing was done during the following PCR enrichment (15 cycles) using custom amplification primers carring the index sequence indicated with 'NNNNNN'. (Primer1: Oligo_Seq AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T, primer3: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT. After two more XP beads purifications (1:1) libraries were quantified. For Illumina flowcell production, samples were equimolarly pooled and distributed on all lanes used for 75bp single read sequencing on Illumina HiSeq 2500.