title: GSM5695534: TAGMe_RNA_GM12878_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: After reaching 70~80 confluence, IMR-90 cells were detached with 0.5% Trypsin and harvested by centrifuge at 300g for 5min. GM12878 cells were harvested at a density around 0.7M to 0.8M/mL by centrifuge at 300g for 5min. Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, then quenched by adding 0.2M Glycine. After washing with ice-cold PBS, cells were suspended in the nuclei isolation buffer with RNase inhibitors and incubated on ice for 1hr. Subsequent to nuclei membrane permineralization, chromatin was digested using 100U DpnII (NEB, R0543L) overnight at 37 ℃ with RNase inhibitors. The next day, DpnII was inactivated, and nuclei suspension was cooled to room temperature and subjected to biotin fill-in. Proximal segments were in situ ligated using T4 DNA ligase (NEB, M0202) supplemented with RNase inhibitors. Then GpC methyltransferase footprint was performed by incubating the nuclei with M.CviPI (NEB, M0227L), and PBS was added to stop the reaction. After that, one-third of the nuclei were separated for RNA isolation using MagMAX™ FFPE DNA/RNA Ultra Kit (Thermo A31881). The total RNA library was prepared using SMARTer® Stranded Total RNA-Seq Kit v2 (TAKARA 634413). The rest of the nuclei were reverse crosslinked, DNA was precipitated and sonicated to average 400bp followed by 1xAMPure beads cleanup. Biotin pulldown was performed using streptavidin beads, DNA on beads was end-repaired, d-A tailed, and ligated with truncated cytosine-methylated X-gene Universal Stubby Adapter (IDT). 0.1%~0.5% unmethylated lambda DNA was spiked-in. Bisulfite conversion was conducted using the EZ DNA Methylation-Gold Kit (D5006). Bisulfite converted DNA library was amplified using truncated TruSeq™–Compatible Indexing Primer and KAPA HiFi Uracil+ ReadyMix (Roche, KK2801), supplemented with MgCl2. Library quality was determined by qPCR and BioAnalyzer 2000 (Agilent Technologies). Pooling of multiplexed sequencing samples, clustering, and sequencing was carried out as recommended by the manufacturer on Illumina HiSeq XTen or NovaSeq S6000 with the 150 paired-end. RNA-seq libraries or Phi-X (20%) were spiked in to overcome the imbalance of  GC ratio of the bisulfite-converted DNA libraries