GSM9821648: IBM4; Homo sapiens; RNA-Seq
For fixed FLEX samples, each samples' FFPE block was cut into several scrolls and prepared into single cell suspensions following 10X Genomics' CG000632 Demonstrated Protocol.  Specifically, the gentleMACS Octo Dissociator for Isolation of Nuclei from FFPE Tissue Sections protocol was employed.  Briefly, scrolls were placed into the gentleMACS C tube and deparaffinized with xylene and rehydrated with graded ethanol washes (100% ethanol, 70% ethanol, 50% ethanol, 100% water), before resuspension in PBS.  Tissue was then dissociated with Liberase TH and the Miltenyi Octo Dissociator.  Nuclei were subsequently filtered through a 30 micron filters and counted with propidium iodided before proceeding to Chromium Fixed RNA Profiling protocols – Probe Hybridization. Library construction was performed following the manufacturer's (10X Genomics) official protocols.  For FFPE samples, the FLEX protocol was utilized and, once cell suspensions were generated from FFPE scrolls for each sample, the protocol Chromium Fixed RNA Profiling – Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture (CG000673) was utilized to hybridize individual probe barcodes per sample.  Once a probe barcode was associated per sample, library construction proceeded following User Guide CG000527 Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples.  Briefly, post-hybridization, the 16 samples containing different probe barcodes were pooled together and unbound probes were washed off.  The samples were then mixed with a Master Mix and loaded into Chromium Chip Q along with gel beads and partitioning oil.  The chip is then loaded into the Chromium X Series instrument to generate the Gel Beads-in-emulsion (GEMs) which are subsequently incubated, amplified, size-selected, indexed, and quantified.  The libraries are standard llumina paired-end constructs, beginning with P5 and ending with P7. The final library includes 16 bp 10x GEM Barcodes (10x Barcode) encoded at the start of TruSeq Read 1 (Read 1T). Finally, the sample index sequences are incorporated as the i5 and i7 index reads.