GSM9701249: Biological replicate 2, technical replicate 2, naive human macrophages; Homo sapiens; RNA-Seq
Cells were sorted via FACS following co-incubation as indicated. Following PBS washing steps to remove excess FBS, samples were lysed into TRI Reagent (Zymo Research, R2071) and then snap frozen and stored at -80°C until RNA extraction was performed. RNA extraction was performed using Direct-zol RNA MiniPrep Plus (Zymo Research, R2071) following manufacturer's instructions. Two biological replicates of each cell type (Eaters, Non-eaters, Uneaten, SW620, and Naive macrophages) were used for RNA sequencing, with each biological replicate consisting of two technical replicates. RNA-seq libraries were prepared using the Single Cell/Low Input RNA Library Prep Kit (NEB, E6420), following the manufacturer's protocol. In brief, 200 ng of total RNA was used as input. First, reverse transcription was performed, followed by 7 cycles of cDNA amplification and clean-up. The cDNA was then fragmented, repaired, ligated with NEBNext adaptors, and enriched using 4-5 cycles of PCR. Libraries were sequenced on a NovaSeq X 10B platform using paired-end 150 bp (PE150) reads.