RNA-seq of homo sapiens: IMR-90
For each cell pellet, total RNA was isolated with RNeasy Plus Mini (Qiagen, Cat. No. 74134) using QIAshredder columns (Qiagen, Cat. No. 79654) for homogenization, then purified with Zymo RNA Clean & Concentrator-5 (Zymo Research, Cat. No. R1015). Isolated RNA was measured for quantity with Quant-iT Ribogreen RNA Assay (Thermo Fisher, Cat. No. R11490) and quality with High Sensitivity RNA ScreenTape and buffer (Agilent, Cat. No. 5067-5579 & 5067-5580). For each RNA sample, a uniquely dual-indexed, Illumina-compatible, double-stranded cDNA whole transcriptome library was synthesized from 1 g of total RNA with Takara Bios SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian kit (Takara Bio, Cat. No. 634874) and SMARTer RNA Unique Dual Index kit (Takara Bio, Cat. No. 634452). Briefly, this library preparation included ribosomal depletion, RNA fragmentation (94 C for 3 min), and a 12-cycle indexing and enrichment PCR. Each library was measured for size with Agilents High Sensitivity D1000 ScreenTape and buffer (Agilent, Cat. No. 5067-5584 & 5067-5603). 1 uL of each library was combined into a non-equimolar pool which was then measured for size with TapeStation and concentration with Roches KAPA SYBR FAST Universal qPCR Kit (Roche, Cat. No. KK4824), diluted to 70 pM, then loaded into an iSeq flow cell cartridge (Illumina, Cat. No. 20031371) with a 1% v/v PhiX Control v3 spike-in (Illumina, Cat. No. FC-110-3001), and sequenced to 101 x 9 x 9 x 101 cycles. Passing filter cluster counts per library from these iSeq data were used to make a re-balanced pool which was subsequently measured for size and concentration, diluted to 750 pM with a 1% v/v PhiX Control v3 spike-in, denatured and further diluted, loaded into a NovaSeq X flow cell lane (Illumina, Cat. No. 20085594), and sequenced at 151 x 8 x 8 x 101 cycles with a final flow cell concentration of 150 pM. Libraries were sequenced to at least 50 M read pairs (or 100 M paired-end reads). The low diversity of the first three bases in Read 1 of these libraries was balanced in both the iSeq and NovaSeq X pools with other library types that had a higher diversity across those bases; otherwise, a 20% PhiX spike-in would have been needed for both pools.