GSM2805046: det_3 RNA-Seq; Mus musculus; RNA-Seq
Total RNA for transcriptome analysis was isolated with miRNeasy Micro Kit (Qiagen) from one six mm2 dishes per sample. The integrity of RNA was checked on Bioanalyzer 2100 (Agilent), and 5μg of RNA with RIN >9 were used for ribosomal depletion using the Ribo-Minus Eukaryote Kit v2 (Invitrogen). Following modifications to the standard RNA-seq Library protocol was performed Next generation sequencing was performed on Ion Torrent Proton sequencing platform (Thermo Fisher) using the Ion Total RNA-Seq Kit v2 (Thermo Fisher) with minor modifications. Total RNA for transcriptome analysis was isolated with miRNeasy Micro Kit (Qiagen) from one six mm 2 dishes per sample. The integrity of RNA was checked on Bioanalyzer 2100 (Agilent), and 5μg of RNA with RIN >9 were used for ribosomal  depletion using the Ribo-Minus Eukaryote Kit v2 (Invitrogen). Following modifications to the standard RNA-seq Library protocol were performed: fragmented RNA was concentrated to 3μl with SpeedVac (Eppendorf) for 10-15min, and all 3μl were used for further steps. The number of amplification cycles was reduced to 8-12 cycles resulting in lower PCR duplication levels without any influence to quality/contribution of obtained reads. To maximize data, recovery libraries were size selected using the LabChipXT system (Perkin Elmer) with an isolation window between 180-270bp (corresponding to 60-150bp insert size). Obtained RNA libraries were quantified on Qubit 2.0 and diluted to 100 pM and used in a final concentration of 10 pM for template preparation on Ion OneTouch2 instrument (Thermo Fisher). For each run on the PI Ion Torrent V2 Chip, 2 RNA libraries were pooled in equimolar ratios to obtain between 37-63M raw reads per sample.