GSM8657469: p22252-s026_Repeat-run_ROk18-LL60-RHv18_D8_BAL_GEX; Macaca mulatta; RNA-Seq
To collect non necropsy BAL, a 14Fr, sterile, single-use pediatric suction catheter (Covidien #37424) was inserted into the trachea and directed into a mainstem bronchi. The catheter was advanced until it was secured into a distal subsegmental bronchus. 35-70 mL of physiological saline was instilled into the bronchus and then manually aspirated back into a syringe to obtain a minimum of 20 mL of lavage fluid and placed into a 50mL conical. If less than 20mL was recovered, an additional 10-15mL was administered again and a second attempt was taken and placed into a second 50mL conical. BAL was then filtered through a 70uM strainer (Falcon #352350). 1mL of BAL fluid and cells were taken to perform viral load testing by adding RNazol and Acetic Acid into a 5mL microcentrifuge tube. After vigorous vortexing, samples were frozen for later RNA extraction using the RNAzol BD kit. After viral loads were taken, BAL was spun at 300 x g for 10 minutes to pellet the cells. BAL supernatant was collected for MSD and plaque assay. BAL cells were then lysed with the ACK lysis buffer for 10 minutes at 37˚C and the reaction was quenched using DPBS. Cells were then spun and counted in the same manner as PBMCs. At least 100,000 cells were set aside for 10x sequence and the remaining cells were divided between the isotype control and stained panel. At necropsy, an additional 100mL of Post Mortem BAL fluid was taken by placing a large sterile irrigation syringe with the plunger removed into the lungs. Approximately 150 mL of sterile 1 x PBS is poured into the lungs via the syringe to infuse both sides. The syringe is then removed and plunger replaced. The syringe goes back into the trachea opening and the lavage fluid is pulled back and dispensed into 50mL conicals. Post mortem BAL sample is processed the same way as non-necropsy through a 70uM filter and lysing. After samples for viral load, supernatant and 10x were taken for pre-mortem samples, the cells for pre and post mortem were combined together for staining. At necropsy, at least 5 million cells were stained for both the isotype and staining panel with any remaining frozen down using the same freezing media as PBMCs. Caudal lung is processed by following the steps indicated in Voix et al 2023 with the following changes. During the 1 hour 37˚C incubation, samples were placed on a 50mL tube rotator at 10 rpm (Thermo Scientific # 888810001). Samples were run at least 6 times on the pre-loaded M_Lung_02_01 to ensure proper blending of the tissue. After, the tissue is strained over 100uM filters (Falcon # 352360) and the digestion reaction is quenched with R10. Cells are then centrifuged at 1100 x g for 10 minutes. The supernatant is removed and cells are washed again with R10 and centrifuged at 1100 x g for 10 minutes. The cells are lysed using 10mL of ACK lysis and incubated at 37˚C for 10 minutes. Lysing was then quenched using DPBS and cells were spun down at 1100 x g for 10 minutes. Cells were then counted using the same manner as PBMCs and aliquoted for 10x sequencing, bulk RNA sequencing and freezing. Cells were used for 10x sequencing and the extra were saved in the same manner as PBMCs.  Spleen was processed in a similar manner to the lung but without the digestion step. Spleen was dissected using blunt scissors and added to C tubes with 5mL of R10. Using the same M_Lung 02_01 protocol run at least 6 times, spleen was blended then passed through a 100uM filter. Due to the nature of the sample, it is then passed through a 70uM filter and then 40uM filter (Falcon #352340) to remove the debris. Cells were then washed using R10 and then spun at 750 x g for 10 minutes to pellet. Supernatant was removed and the cells were washed with DPBS then centrifuged at 750 x g for 10 minutes. Cells were lysed with 20mL of ACK for 10 minutes. The lysing reaction was quenched with DPBS and cells were pelleted following the same speed and time. Cells were counted and frozen for later use. Single cell suspensions were prepared and loaded onto the 10X Genomics Chromium Controller in the BSL3 facility using the Chromium NextGEM Single Cell 5' Library & Gel Bead kit to capture individual cells and barcoded gel beads within droplets (113). The libraries were prepared according to manufacturer instructions, including the preparation of feature barcode libraries for hashtag detection. They were then sequenced on an Illumina NovaSeq 6000 with a paired-end 26x91 configuration targeting a depth of 50,000 reads per cell. Cell Ranger software was used to perform demultiplexing of cellular transcript data, as well as mapping and annotation of UMIs and transcripts for downstream data analysis.