GSM9618733: PUER cells, GCSF, OHT, 24h, Rep3; Mus musculus; RNA-Seq
Total RNA was extracted on a Bio-Mek FXP liquid handling workstation (Beckman Coulter) using the RNAdvance Tissue total RNA isolation kit (Beckman Coulter, A32649) from 3 replicates in a 96-well format following the manufacturer's protocol. Genomic DNA contamination was assessed by reverse transcribing the RNA with and without reverse-transcriptase and detecting GAPDH using qPCR. The number of additional cycles required to reach a threshold (ΔCt) was utilized to assess the fraction of genomic DNA (2- ΔCt) and only samples with less than 1% genomic DNA were utilized for library preparation. The quality of the RNA was assessed by capillary gel electrophoresis on the Agilent 2100 Bioanalyzer using the Eukaryote Total RNA Nano kit (Agilent, 5067-1511). Only samples with RNA integrity numbers (RIN) greater than or equal to 9.5 were utilized, although there was only one sample with a RIN of 9.5 and the median RIN was 9.9. The concentration of RNA was determined using the Qubit fluorometer and the Qubit RNA High Sensitivity kit (Invitrogen, Q32855). With the exception of 8 samples with lower RNA yield, the samples were standardized to a mass of 1,875 ng in a 25µl volume. The samples with lower yield were standardized to a mass of 1,437.5 ng in a 25µl volume. 3.75µl or 2.88µl of a 1:100 dilution of the External RNA Control Consortium (ERCC) ExFold RNA Spike-In mix (Invitrogen, 4456739) was added to the high- and low-yielding samples respectively. Illumina libraries were prepared by Novogene Corporation Inc. (Chula Vista, CA) using the NEB Ultra II RNA Library Prep Kit for Illumina according to manufacturer protocols. The libraries were sequenced on an Illumina Novaseq 6000 S2 2 X 150 bp flow cell to an average depth of 29.24 X 106 raw reads per sample for a total of 4.82 X 109 reads. The sequencing provider filtered the reads to remove ones containing Illumina adaptors, or more than 10% indeterminate bases (“N”s), or having more than half bases with a phred score below 5. The remaining “clean” reads, representing 95.52% of raw reads, were used for processing.