GSM8887597: 02_WT2_RNAseq; Mus musculus; RNA-Seq
Total RNA was extracted by TRIzol (Invitrogen), and then purification using PureLink RNA Mini Kit (Cat No. 12183018A; Invitrogen) with DNase treatment (QIAGEN). 2100 Expert (Agilent) was used for RNA quality and quantity assessment. RIN (the RNA Integrity Number) of all samples were above 9.0. Library was prepared for splenocytes using NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400) and NEBNext Ultra II Directional RNA Library Prep Kit from Illumina (NEB #E7760). For PCR amplification (13 cycles) NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, NEB, E6440) were used. Dual index (101x8x8x101) pair-end sequencing of the final 750 pM library was performed using the Illumina NextSeq2000 P2 Reagents (200cycles) on the Illumina NextSeq2000 instrument. The total number of reads was 550M. Biotinylated, target-specific oligos combined with Ribo-Zero ribosomal RNA (rRNA) removal beads were used for the removal of rRNA library construction for splenocytes. Illumina TruSeq Stranded Total RNA Library Preparation kit and paired-end sequencing of 6 pooled RNA-seq samples was performed on HiSeq. Following RNA fragmentation and cleavage, small pieces are converted to complementary DNA (cDNA) with reverse transcriptase and random primers and, consequently 2nd strand cDNA synthesis with DNA Polymerase I and RNase H. A standard Illumina library prep with end-repair, adapter ligation and PCR amplification being performed to give you a sequencing ready library from the double-strand cDNA as input. After that, purified product was quantified using qPCR followed by cluster generation and pair-end sequencing on HiSeq.