GSM4878493: 607_N1_Kdm6a; Mus musculus; RNA-Seq
RNA was extraced with the RNeasy Mini Kit (Qiagen) according to the manufacturer`s protocol. [1] Single end data: 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 ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding beads in a ratio of 1:1, followed by an index PCR (15 cycles) using Illumina compatible index primer. After double XP beads purifications (with beads added in a ratio 1:1) libraries were quantified on a Fragment Analyzer run with a NGS Assay Kit (Agilent) and loaded on a HiSeq 3000 flowcell with 50 cycles single end seqeuncing by pooling the samples in respect of their molarity aiming at 30 mio. reads per sample. [2] paired-end data: Illumina TruSeq mRNA stranded