GSM7805924: Delaminated sensory epithelium, Treated, Biol rep 1; Homo sapiens; RNA-Seq
Sample preparation and cell capture were performed using the 10x Genomics Chromium Single Cell 3' v3 Reagent Kits user guide. Briefly, two washes were done to exchange sample buffer for one recommended for loading on the Chromium Controller. Samples were spun at 0.3G for 6 min at 4oC and resuspended in a solution of 250 µL RPMI-1640 Medium with 0.04% BSA and 5U/µL RNase Inhibitor. Cell viability was assessed via Trypan Blue (Thermo Fisher) and using a haemocytometer (Thermo Fisher). A second spin was performed under identical conditions, and cells were resuspended in 55 µL final volume. Following counting, cell suspension (46.6 µL) was loaded onto the Chromium Single Cell B Chip Kit. After droplet generation, samples were transferred onto a pre-chilled 96-well plate (Eppendorf), heat-sealed, and incubated overnight in a Veriti 96-well thermos cycler (Thermo Fisher) for the reverse transcriptase reaction. Following this reaction, cDNA was recovered using the Recovery Agent provided by 10x Genomics and subsequently cleaned up using a Silane DynaBead (Thermo Fisher) as outlined by the user guide The molarity of each library was calculated based on library size as measured by BioAnalyzer (Agilent Technologies), qPCR amplification data (Sigma) or Illumina MiSeq clustering data (Illumina). Briefly, samples were normalized, then diluted using elution buffer (Qiagen) with 0.1% Tween® 20 (Sigma-Aldrich), and each pool denaturated at r.t.. Library pools were further diluted before being diluted to a final loading concentration. 100 µL from each pool were loaded into each well of an 8-well strip tube and loaded onto a cBot (Illumina) for cluster generation. All samples were sequenced with the following run parameters: read 1 - 28 cycles, read 2 - 91 cycles, and index 1 - 8 cycles. A median sequencing depth of 60,000 reads/cell was targeted for each sample.