GSM9140022: old_2; Mus musculus; ATAC-seq
Whole hearts (both ventricles and atriums) were isolated from 3 young (14 weeks) and 3 old (98 weeks) mice. Washed in PBS and snap-frozen in liquid nitrogen. Hearts from each condition were stored at -80 C. 4 hearts (2 young and 2 old) were mechanically disrupted, isolated nuclei were stained for DAPI (nuclear marker) and PCM1 (cardiomyocyte nuclear envelope marker). Afterwards nuclei were FAC-Sorted based on forward and side scatters and DAPI signal to get nuclei suspension. FAC-Sorted nuclei were used for scATAC-seq, using the 10x Chromium kit. Nuclei were tagmented by Tn5 transposase (cut fragments stay inside the nuclei) and then encapsulated into droplets with barcoded beads. Droplets contain compounds, which release Tn5-cut DNA fragments from the nuclei. Barcoded beads have primers attached to their surface. These primers contain unique bead-barcode, as well as complimentary sequence to adapters, inserted by Tn5 during tagmentation. Therefore, Tn5-cut fragments attach to the bead. On-the-beads PCR enriches attached fragments, incorporating unique bead- barcodes into library. Fragments are released from the beads, additionally enriched (with universal primers) by PCR. The final libraries were cleaned with SPRI magnetic beads and size-selected for 200-1000bp library size. Concentration was assessed by Qubit and library size distribution was assessed by PerkinElmer LabChip.