GSM9405337: CITE-seq, sample47, GEX; Homo sapiens; OTHER
Human single cell isolation for CITE-seq Fresh human coronary arteries from explanted hearts at the time of transplantation or donors declared dead after cardiac death (DCD) or brain death (DBD) at Washington University School of Medicine and Mid America Transplant Service were harvested, perfused with cardioplegia, and transported on ice. Starting at the coronary sinus, the left main, left anterior descending, and left circumflex arteries were dissected removing any epicardial fat while preserving the adventitia. Sections were partitioned for histopathology, spatial transcriptomics, single cell isolation, and flash frozen. Cells were isolated as before (17). Briefly: Coronaries were minced using a razor blade on ice and transferred to a 15 mL conical tube containing 3 mL DMEM with 170 µL Collagenase IV (250 U mL-1 final concentration), 35 µL DNAse1 (60 U mL-1), and 75 µL Hyaluronidase (60 U mL-1, all purchased from Sigma Aldrich) and incubated at 37 °C for 45 min with agitation. After 45 min, the digestion reaction was quenched with 6 mL of HBB buffer (2% FBS and 0.2% BSA in HBSS), filtered through 40 µm filters into a 50 mL conical tube and transferred back into a 15 mL conical tube to obtain tighter pellets. Samples were then centrifuged at 4°C, 1,200 rpm for 5 min, and the supernatant was discarded. The pellets were resuspended in 1 mL ACK Lysis buffer (#A10492-01, Gibco) and incubated at room temperature for 5 min followed by the addition of 9 mL DMEM and centrifugation at 4°C, 1,200 rpm for  5 min. Supernatant was discarded and the pellet was resuspended in 2 mL FACS buffer (2% FBS and 2 mM EDTA in calcium/magnesium free PBS), and centrifugation was repeated in above conditions and supernatant aspirated. The TotalSeq A 277 panel (BioLegend) antibody cocktail was resuspended in 100 µL of FACS buffer and 1 µL each of custom oligo-labeled FAP (Amgen) and LRRC15 (Amgen) were added.  The combined 102 µL were used to resuspend the pellet with the addition of 1 µL of DRAQ5 (#564907, Thermo Fisher Scientific) and incubated on ice for 30 min. Solution was washed with FACS buffer three times following same centrifugation as above and then resuspended in 500 µL of FACS buffer and 1 µL DAPI (#564907, BD Biosciences), then filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+/DAPI- events were collected in 300 µL cell resuspension buffer (0.04% BSA in PBS). The collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells µL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. CITE-seq Library Preparation Collected cells were processed using the single Cell 3' Kit v 3.1 (#1000268, 10x Genomics). 10,000 cells were loaded onto ChipG (#1000121) for GEM generation. Reverse transcription, barcoding, and complementary DNA amplification of the RNA and ADT tags were performed as recommended in the 3' v3.1 chromium protocol. Custom conjugated FAP antibodies were generated using TotalSeqA barcodes by Biolegend. sequenced on a NovaSeq6000. CITEseq libraries were generated using TotalSeqA reagents and Chromium Next GEM Single Cell 3ʹ Kit v3.1 reagents (#1000268, 10x Genomics) following the 10x Genomics protocol (Document CG000185) modified to capture the TotalSeqA barcodes.  Briefly, during cDNA amplification, 1 µl of 0.2 µM ADT Additive Primer (CCTTGGCACCCGAGAATT*C*C) was added to the reaction.   5 µl of purified ADT fraction was indexed with SI-PCR primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC*T*C) and RPI Primer (CAAGCAGAAGACGGCATACGAGAT[i7index]GTGACTGGAGTTCCTTGGCACCCGAGAATTC*C*A) at a final concentration of 2.5 µM each using 2x Kapa Hifi Master Mix. Libraries were sequenced on a NovaSeq 6000.