GSM9066325: Gene expression; Homo sapiens; RNA-Seq
Cultured cells from 3 female donors were washed with PBS, then incubated on ice for 5min with 100ul hypotonic buffer (10mM Tris-HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP40). After 5min incubation, wells were loosely scraped with a pipette tip and 200ul of wash buffer (PBS 1X, 1% BSA, 0.2U/ul Protector RNAse Inhibitor) and counted using a Cellometer K2 with AO/PI live/dead reagent. Technical replicates "well A" and "well B" for each donor were sequenced and combined for downstream analysis. Nuclei tagging was done using the MULTIseq methodology in tandem with the Curiox washing system (Curiox Biosystems). In brief, anchor lipid-oligo and Barcoding oligos specific to each sample were incubated together then added to the respective samples to get incorporated into the nuclei membrane. A Co-anchor lipid-oligo was added to the mix to stabilize the tagging before transferring the samples to individual wells of a Curiox washing strip. Nuclei were incubated 40min on ice to bind oligos and allow nuclei to settle in the wells. Nuclei were washed 25x at flow rate of 1ul/s with PBS + 1% BSA prior to recovery, counting and pooling in equal proportions.