title: GSM9458831: RM1-095D4; Homo sapiens; RNA-Seq
library_construction_protocol: CD66b+ cells were isolated and total RNA isolated using RNeasy Mini kit (Qiagen 74104) following manufacturers' recommendations.  RNA was quantified using Qubit RNA Broad Range Assay Kit (Ref# Q10210; Invitrogen, MA) and RNA integrity was assessed using RNA ScreenTape kit (Ref# 5067-5576) on TapeStation 4200 (Agilent). Illumina Stranded mRNA Prep and Ligation kit (Ref# 20040534; Illumina, CA) were used to prepare sample libraries following the manufacturer's recommendations. Very briefly, 300 ng/uL high quality RNA was captured and purified, followed by reverse transcription to convert the captured mRNA into first and second strands of cDNA. The resulting double stranded cDNA molecules were then enzymatically fragmented in preparation for unique dual indexing using IDT for Illumina RNA UD Set Ligation Indexes (Ref# 20091655; Illumina, CA). The ligated samples were subsequently enriched through PCR amplification on Bio-Rad Thermal cycler (T100; Bio-Rad, CA) and purified using AMPure XP magnetic beads (Ref# A63881; Beckman Coulter, CA). The average fragment length of the library insert was measured to be ~300–400 bp using the dsDNA 1000 kit (Ref# 5067-5582) on Agilent TapeStation. Optimized libraries were pooled to a final concentration of 150pM. The libraries were sequenced on NovaSeq X Plus Platform (Illumina, CA) at UF's Interdisciplinary Center for Biotechnology Research (ICBR).