DOGMA-seq of Healthy Adult Human: Sample Pool_4
"Cryopreserved PBMCs were thawed, washed, and stained with barcoded hashing and phenotyping TotalSeqA antibodies (BioLegend) as previously described for DOGMA-seq (PMID: 34083792). Stained cells were fixed in 0.1% formaldehyde for 5 min at room temperature and quenched with glycine solution to a final concentration of 0.125 M before washing twice in PBS via centrifugation at 400 g, 5 min, 4oC. Cells were subsequently treated with the lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 1% BSA) for 3 min on ice, followed by adding 1 ml of chilled wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA). Cells were harvested and diluted in 1x Diluted Nuclei buffer (10x Genomics) and filtered through a 40 um Flowmi cell strainer before counting using Trypan Blue and a Countess II FL Automated Cell Counter. From there on, the 10x Multiome protocol was followed with these two modifications: 1. During preamplification PCR, 1 ul of 0.2 uM additive primer (ADT and HTO) was added. ADT add = 5-CCTTGGCACCCGAGAATT*C*C-3 HTO add = 5-GTGACTGGAGTTCAGACGTGTGC*T*C-3. 2. 35% of the pre-amplified sample was used to amplify and index protein tags."