ANG141
Library preparation was performed per the manufacturer's instructions, with the following modifications: 25 pg of External RNA Controls Consortium Spike-in mix (ERCCS, Thermo-Fisher) was added to each sample prior to RNA fragmentation; the input RNA mixture was fragmented for 8 min at 94C prior to reverse transcription; and a total of 14 cycles of PCR with dual-indexed TruSeq adapters was applied to amplify the resulting individual libraries. An initial equivolume library pool was generated, and the quality and quantity of that pool was assessed via electrophoresis (High-Sensitivity DNA Kit and Agilent Bioanalyzer; Agilent Technologies, Santa Clara, CA, USA), real-time quantitative polymerase chain reaction (qPCR) (KAPA Library Quantification Kit; Kapa Biosystems, Wilmington, MA, USA), and small-scale sequencing (2 x146 bp) on an iSeq platform (Illumina, San Diego, CA, US). Subsequent equimolar pooling of individual libraries from each plate was performed prior to performing large-scale paired-end sequencing (2  146 bp) run on the Illumina NovaSeq sequencing system (Illumina, San Diego, CA, USA). The pipeline used to separate the sequencing output of the individual libraries into FASTQ files of 146bp paired-end reads is available on GitHub at https://github.com/czbiohub/utilities.