title: Illumina NovaSeq 6000 paired-end Sequencing of KAS23061420 (KAE23676470)
design: To assess the integrity of total RNA, samples were run on a TapeStation RNA ScreenTape (Agilent Technology; Santa Clara, CA, USA). Only high-quality RNA preparations with RIN greater than 7.0 were used for RNA library construction. A library was independently prepared using 1 microgram of total RNA for each sample and Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina; San Diego, CA, USA). The first step in the workflow involved purifying polyA containing mRNA molecules using polyT attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random primers, followed by second strand cDNA synthesis using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments then underwent an end repair process, addition of a single A base, and ligation of adapters. Products were then purified and enriched by PCR to create the final cDNA library. Libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms (Kapa Biosystems; Wilmington, MA, USA) and qualified using the TapeStation D1000 ScreenTape (Agilent).
library_construction_protocol: To assess the integrity of total RNA, samples were run on a TapeStation RNA ScreenTape Agilent Technology; Santa Clara, CA, USA. Only high-quality RNA preparations with RIN greater than 7.0 were used for RNA library construction. A library was independently prepared using 1 microgram of total RNA for each sample and Illumina TruSeq Stranded mRNA Sample Prep Kit Illumina; San Diego, CA, USA. The first step in the workflow involved purifying polyA containing mRNA molecules using polyT attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase Invitrogen and random primers, followed by second strand cDNA synthesis using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments then underwent an end repair process, addition of a single 'A'?base, and ligation of adapters. Products were then purified and enriched by PCR to create the final cDNA library. Libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms Kapa Biosystems; Wilmington, MA, USA and qualified using the TapeStation D1000 ScreenTape Agilent.