NextSeq 500 paired end sequencing; scRNA-seq analysis of cell subpopulations of the bronchoalveolar lavage fluid of healthy dogs
scRNA-seq analysis of cell subpopulations of the bronchoalveolar lavage fluid of healthy dogs
Bronchoalveolar lavage fluid (BALF) were obtained under anesthesia with butorphanol at 0.2 mg/kg (Butomidor®, Richter Pharma AG, Wels, Austria) as premedication and propofol (Diprivan®, Asen Pharma Trading Limited, Dublin, Irland) infusion on demand. Dogs were not intubated for the procedure. A bronchoscope (FUJINON© Paediatric Video-Bronchoscope EB-530S), cleaned and disinfected using the washer-disinfector Serie 4 (Soluscope®, Aubagne, France), was inserted into the bronchi until the extremity was wedged. Three to four mL/kg of a sterile NaCl 0.9% solution divided into 3 aliquots were instilled through the endoscope channel into the lung (2 aliquots were obtained in the right diaphragmatic lobe and one in the left diaphragmatic lobe) and directly reabsorbed by gentle suction into the same sterile recipient. BALF was then directly transferred on ice to the GIGA laboratory of Cellular and Molecular Immunology. BALFs were filtered to remove mucus and total cell count was assessed using a haemocytometer and Türk coloration. BALFs were then centrifuged at 400 g for 7 minutes and the pellet resuspended in phosphate-buffered saline solution (GibcoTM 1x DPBS, Cat.14190-169) to obtain a cell concentration around 1,000 cells/µL. A second filtration through a cell strainer (BD Falcon™, Biosciences, USA, Cat.352350) was performed to remove any remaining cell debris and large clumps and cells were again counted with Trypan blue staining to assess cell viability considered as acceptable above 70%. The volume of the cell suspension was then adjusted to obtain a final cell concentration between 500 and 1000 cells/µL suspended in phosphate-buffered saline solution containing 0.04% (w/v) bovine serum albumin.    Cellular suspensions were loaded on the Chromium™ Controller (10x Genomics, Pleasanton, CA, USA) in order to generate Gel Bead-In-EMulsions (GEMs). Sequencing libraries were performed using Chromium™ Single Cell 3' Reagent Kits v2 (10x Genomics, Pleasanton, CA, USA) according to manufacturer's instructions. GEM-RT was performed in a Veriti© 96-Well Thermal Cycler (Fisher Scientific, Merelbeke, Belgium). After RT, GEMs were broken and the cDNAs were cleaned up with DynaBeads MyOne Silane Beads (Fisher Scientific, Merelbeke, Belgium). cDNAs were then amplified in a Veriti© 96-Well Thermal Cycler. According to the expected cell recovery (based on a 60% recovery of total loaded cells), number of amplification cycles was set to 14. Amplified cDNA products were cleaned up using SPRIselect Reagent kit (Beckman Coulter, CA, USA), after what purified cDNAs were quality controlled and quantified using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser (Agilent, CA, USA). Illumina's P5, P7 and Read2 primers, as well as Sample Index were then added to generate sequencing libraries following Chromium™ Single Cell 3' Reagent Kits v2 protocol. Steps were as follows: (1) enzymatic fragmentation, end repair and A-tailing, (2) Double Sided Size Selection using SPRIselect reagent, (3) adaptor ligation, (4) post ligation cleanup with SPRIselect reagent, (5) Sample index PCR (number of cycles set to 14) and (6) Double Sided Size Selection using SPRIselect reagent. The barcoded sequencing libraries were quality controlled using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser and quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms).