Nextseq 500 550 high output flow cell v2.5

Libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit with the IDT for Illumina Unique Dual Index adapters following manufacturer’s recommendations. Completed libraries were QC’d and quantified using a combination of Qubit dsDNA HS and Agilent 4200 TapeStation High Sensitivity DNA 1000 assays. The libraries were pooled in equimolar amounts and the pool quantified using the Kapa Biosystems Illumina Library Quantification qPCR kit. This pool was loaded onto an Illumina NextSeq 500/550 High Output flow cell (v2.5) and sequencing performed in a 1x75 bp single read format using a NextSeq 500/550 High Output 75 cycle reagent kit (v2.5). Base calling was done by Illumina Real Time Analysis (RTA) v2.4.11 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1. Sequence data have been accessioned in NCBI’s SRA under the BioProject PRJNA704083.

The DNA and protein tag libraries were run on a High Sensitivity D1000 ScreenTape instrument (Agilent Technologies, 5067-5584) with the Agilent 4200 TapeStation System to evaluate DNA quality. Libraries were quantified by a fluorometer (Qubit 4.0, Invitrogen) and sequenced on Illumina next-generation sequencing platforms with a 20% spike-in of PhiX control DNA (Illumina, no. FC-110-3002). DNA and protein tag libraries were sequenced separately on a NextSeq 500 instrument (Illumina), using the NextSeq 500/550 High Output flow cell v2.5 (Illumina, no. 20022408) and the NextSeq 500/550 High Output Kit v2.5 (300 cycles; Illumina, no. 20024908) in 2 × 150-bp paired-end runs.