GSM6617296: Aortic CD45+, WT and myeloid JAK2-V617F, HTO; Mus musculus; OTHER
Jak2WT (n=5) and Jak2V617F MyC (n=5) mice under isoflurane anesthesia received 2µg of anti-CD45.2-APC (clone 104, BioLegend) i.v. 5 minutes before sacrifice to label circulating immune cells and were killed by cervical dislocation. Upon collection of the aorta using a stereomicroscope, aneurysm development was macroscopically detected in 2 Jak2V617F MyC mice. Aortas were collected and enzymatically digested in RPMI medium containing 450U/ml collagenase I (Sigma-Aldrich C0130), 125U/ml collagenase XI (Sigma-Aldrich C7657), 60U/ml Hyaluronidase (Sigma-Aldrich H3506) and 60U/ml DNAse1 (Roche #11284932001) for 45 minutes at 37°C with agitation, washed in PBS+1% FCS and passed through a 70µm cell strainer. After washing in PBS+1%FCS, aortic cells were resuspended in erythrocyte lysis buffer (ACK buffer) for 5 minutes and washed again with PBS+1% FCS. Cells were plated in round bottom 96 well plates, and incubated for 10 minutes on ice with anti-CD16/32 (BioLegend, Clone 93, 10µg/ml in PBS+1% FCS) to block unspecific binding of antibodies to Fc receptors. Cells were subsequently labeled in a final volume of 60µl for 25 minutes at 4°C with a mix containing CITE-seq antibodies (TotalSeq-A, BioLegend), 1:1000 Fixable Viability Dye e780 (ThermoFisher Scientific 65-0865-14), 2µg/ml anti-CD45.2 BV421 (Jak2WT samples) or 2µg/ml anti-CD45.2 Alexa488 (Jak2V617F samples). Each unique sample was labeled with a specific Hashtag antibody for cell hashing (Hashtag 1 to 5: Jak2WT aortas, Hashtag 7 and 9: Jak2V617F MyC with aneurysm; Hashtag 6, 8 and 10: Jak2V617F MyC without aneurysm; final concentration 1:100, BioLegend TotalSeq-A). After labeling, cells were washed twice in PBS+1% FCS and pooled. Viable leukocytes were sorted with exclusion of contaminating circulating leukocytes (CD45.2-APC+) using a BD FACS Aria III with a 100µm nozzle. The amount of sorted cells was adjusted so that cells from Jak2WT (CD45.2-BV421+) and from Jak2V617F aortas (CD45.2-Alexa488+) each represented ~50% of the total sorted cells. Cells were sorted in PBS supplemented with 1% PBS, and washed twice post-sort in PBS+0.04% UltraPure BSA (ThermoFisher AM2616). Cells were counted in Trypan blue to assess viability (>80%). 20,000 cells were loaded at a concentration of 500 cells/µl in the 10x Genomics Chromium (Single Cell 3' v3 reagents, 10x Genomics) and scRNA-seq, ADT and HTO libraries were prepared and sequenced. Library was prepared using Chromium Single Cell 3ʹ v2 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3' v2 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags. SPRI bead size selection was performed in order to separate both the mRNA-derived cDNA (>300bp) and the antibody-derived tagged cDNAs (180bp). For the mRNA derived cDNA library preparation, we further proceeded according to the manufacturer's instructions for Single Cell 3' v2 protocol (10x Genomics). For antibody-derived tagged library, we used the KAPA HiFI HotStart Library Amplification Kit (Roche) with the following primers and amplification program: -CITE-seq library: amplification was performed at 95°C for 3' followed by ten cycles at 95°C f¬¬or 20''; 60°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3' Small RNA RPI1 primer as i7 primer (Illumina). -HASH-seq library: amplification was performed at 95°C for 3' followed by ten cycles 95°C for 20''; 64°C for 30''; 72°C for 20'' and final elongation 72°C for 5' with 5'-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC-3' TruSeq D701_s primer as i7 primer (Illumina). The following 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3' SI-PCR primer as i5 primer (Illumina) was used for both CITE- and HASH- seq library amplification. Following the final bead purification, all three libraries were pooled as 80% mRNA library, 10% ADT library and 10% HTO library before sequencing.