GSM9597751: R004-3; Homo sapiens; RNA-Seq
Total RNA was isolated using AllPrep DNA/RNA Mini Kit (80204, Qiagen, Germantown, MD, USA) following the manufacturer's instruction with modifications to capture small RNAs simultaneously. Stranded total RNA-sequencing was performed on the Illumina NovaSeq X Plus Sequencing System in the Genomic Medicine Center at Children's Mercy. Quality control was completed with the RNA Screen Tape Assay kit (5067-5576, Agilent Technologies) on the Agilent TapeStation 4200. Total RNA (500ng) was processed using the Illumina TruSeq Stranded Total RNA Library Prep Gold (20020598, Illumina) in the following steps: i) Ribosomal RNA (rRNA) depletion using using biotinylated, target-specific oligos combined with Ribo-Zero rRNA, ii) fragmentation, iii) reverse transcription/first strand synthesis, iv) Second strand synthesis v) A-Tailing, vi) Unique Dual Index (UDI) adaptor ligation, and vii) library amplification. Library validation was performed with the D1000 Screen Tape Assay kit (5067-5582, Agilent) on the Agilent Tape Station 4200. Library concentrations were determined with a Qubit 4 Fluorometer (Thermo Fisher Scientific). Libraries were pooled based on equal molar amounts and the multiplexed pool was quantified using a Qubit 4 Fluorometer and sized using the D1000 Screen Tape Assay kit. The RNA-Seq library pool was adjusted to 3 nM for multiplexed sequencing. Pooled libraries were denatured with 0.2 N NaOH (0.04N final concentration), and diluted to 90 pM. Onboard clonal clustering of the patterned flow cell was performed using the NovaSeq X Plus 25B (300 cycle, 20104706, Illumina). A 2x151 cycle sequencing profile with dual index reads was completed using the following sequence profile: Read 1 – 151 cycles x Index Read 1 – 17 cycles x Index Read 2 – 8 cycles x Read 2 – 151 cycles.