GSM9048002: NL_02-06-0003-7; Homo sapiens; RNA-Seq
Nasal lavage samples were collected using a previously established protocol. Briefly, 240ml of sterile sodium bicarbonate buffered normal saline in a sinus rinse bottle (NeilMed Sinus Rinse) was used to rinse the sinuses and collect lavage material. After collection, the lavage was centrifuged, resuspended, strained and homogenized. The sample was split into 3 aliquots to be used for 1) histology assessed by cytospin and manual differentials, 2) bulk RNA-sequencing by pelleting and preserving in 1ml of RNAprotect Saliva Reagent (Qiagen), and 3) single cell RNA-sequencing by pelleting and immediately fixing the cell pellet (within 60 minutes of sample collection) using the 10X Chromium Flex RNA protocol (10x Genomics), according to the manufacturer's instructions, using a 1 hour fixation. Fixed samples were shipped to a central facility where they were thawed and hybridized overnight to one of four probe sets (10x Genomics). Samples were pooled in groups of four, washed, and loaded onto the Chromium X (10x Genomics) for probe capture. Sequencing libraries were generated using the Chromium Fixed RNA Profiling Reagent Kit for Multiplexed Samples. Gene expression libraries were pooled and treated with Illumina Free Adapter Blocking Reagent (Illumina). Sequencing was carried out on a NextSeq 2000 sequencer (Illumina) using NextSeq P3 flowcells (Illumina) with a target depth of 10,000 reads/cell.