GSM7732514: Paired_tag_islet_H3K27ac_0week_B7_sub_library_rep1_RNA#01; Mus musculus; OTHER
Single-cell suspension were prepared from the pancreatic islet, Spin-down cells at 300 g for 5 mins, resuspended the cells in 1 mL 0.2% NIB-RPI(1 mL 0.2% NIB, 20 µL Protease Inhibitor (50X), 25 µL SUPERase IN and 12.5 µL RNaseOUT). Sit on ice for 5 min.Count the nuclei using cell counter. Libraries were constructed by Paired-tag workflow, briefly: First, permeabilized nuclei were incubated with antibodies (H3K27ac, Cell Signaling #8173S, H3K4me1, Cell Signaling #5326S) 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: 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 to give rise DNA and RNA libraries for sequencing