GSM5667911: Ageing, H3K9me3, Paired-Tag, DNA#10; Mus musculus; OTHER
Single-cell suspension were prepared from douncing of the frozen tissues, in Doucing Buffer with Protease/RNase Inhibitor cocktail (DBI: 0.25 M sucrose [Sigma, S7903], 25 mM KCl [Sigma, P9333], 5 mM MgCl2, 10 mM Tris-HCl pH 7.4, 1 mM DTT [Sigma, D9779], 1X Protease Inhibitor, 0.5 U/µL RNase OUT and 0.5 U/µL SUPERase Inhibitor) supplemented with 0.1% Triton-X 100 (Sigma T9284). The cell suspension was then filtered by 30 µm Cell-Tric (Sysmex) and spun-down for 10 min, 1,000 g at 4 °C. After washing the cell pellets with DBI and spun-down again, NIB with 0.2% IGEPAL CA-630 were added to resuspend the nuclei pellets and rotate for 10 min at 4 °C. The nuclei were counted by BioRad TC20 cell counter and proceed to Paired-tag experiments immediately.Libraries were constructed by Paired-tag workflow, briefly: First, permeabilized nuclei were incubated with antibodies targeting chromatin regions of interest to trigger the targeted binding of protein A-fused Tn5. Tagmentation reaction and reverse transcription (RT) were then sequentially performed: specifically, reactions were carried out in 12 different wells, with the same set of barcodes in transposase adaptors and RT primers, to label different samples or replicates (1st round of barcodes). Next, a ligation-based combinatorial barcoding strategy was used to introduce the 2nd and 3rd rounds of barcodes to nuclei, by sequential attaching well-specific DNA barcodes from 96-well plates to the 5'-end of both DNA fragments and cDNA from RT. Lastly, barcoded nuclei were divided into sub-libraries and lysed, the DNA/cDNA was purified and subjected to an optimized “amplify-and-split” strategy based on that we previously developed, to give rise DNA and RNA libraries for sequencing