GSM9363783: PA1-Dicer mutation S1344L homozygous; Homo sapiens; RNA-Seq
The Qiagen miRNA easy kit was used to isolate the total RNA fraction 500ng of total-RNA was added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. Fragmentation time was dependent upon the quality of each total RNA sample. cDNA was end-repaired before ligation of sequencing adapters, and adapter-ligated libraries were enriched by 10 cycles of PCR using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB, #E6440 or E6442). Final libraries had an average peak size of 332bp.