GSM5229375: MNaseseq_SPT6_dTAG7_1h_1; Homo sapiens; MNase-Seq
MNase-seq: the cells were washed with cold DPBS, resuspended in hypotonic buffer (10 mM HEPES pH 7.4, 2 mM MgCl2 and 0.5% NP40; supplemented with protease inhibitor cocktail (Sigma)) on ice and incubated 5 minutes. We added 200 gel units MNAse (NEB), heated up to 37°C, added CaCl2 to a final concentration of 2 mM and incubated at 37°C for 4 minutes before adding two volumes of 2xStop solution containing 20 mM Tris–HCl, pH 8, 4 mM MgCl2, 300 mM NaCl, 20 mM EDTA, 3 mM EGTA, 2% SDS, 0.2 μg/μL RNase A, and 2 pg/mL D.melanogaster mononucleosome spike-in DNA. The samples were then incubated at 37°C for 30 minutes, followed by addition of 200 µg proteinase K (Thermo Scientific) and 15 minutes of incubation at 55°C. The DNA fragments were then purified by phenol:chloroform:IAA extraction and precipitation with isopropanol. 5cap-seq: We spiked the samples with lysates containing 10 million D.melanogaster S2 cells and isolated RNA according to the manufacturers protocol (Qiazol). MNase-seq: RNA libraries were prepared for sequencing using standard NEB protocols (NEBNext Ultra II DNA Library Prep Kit for Illumina). 5cap-seq: ligation of 5' DNA/RNA adaptor with an 8 nt unique molecular identifier, framgentation and random priming to generate the first strand, generation of second strand with biotinylated primer, caputure on strapavidin beads, ends-repair and A-tailing followed by adaptor ligation and amplification with primers with multiplexing barcodes.