An article published in GenomeBiology presents scTAM-seq: a high-throughput, high-confidence method for analyzing DNA methylation at single-CpG resolution across thousands of single cells.
Peripheral blood samples were obtained from two healthy donors from the. Frozen bone marrow mononuclear cells were purchased from StemExpress® and correspond to a 22-year-old healthy male donor.Briefly, peripheral blood was collected and stored at room temperature . Within 24 h after sample collection, B-cell subpopulations were isolated using the RosetteSeqTM Human B Cell Enrichment Cocktail followed by a Ficoll®-Paque Premium density gradient centrifugation.
A total of 120,000–140,000 cells were loaded into a Tapestri microfluidics cartridge. Upon encapsulation, cells were lysed. To obtain the digested samples, the DNAm-sensitive endonuclease was added to the barcoding master mix as follows: 288 μl of Tapestri Barcoding Mix V2, 5 μl of highly concentrated HhaI enzyme , while keeping the remaining reagents as stated in the Tapestri protocol .
: Fig. S4. If more than one undigested sample is generated, we suggest to use as a control the undigested sample that shows the highest correlation with each digested sample, respectively.The Tapestri User Guide V2 was followed to prepare the libraries, with a few modifications listed here. Briefly, PCR products were retrieved from individual droplets and purified with 0.7X Ampure XP beads , to split the scTAM-seq library bound to the beads from the surface-protein library in the supernatant.
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