GSM8529558: Endoderm differentiation, 5 histone marks and IgG control, 1 round, scMTR-seq, sublibrary7, RNA; Homo sapiens; OTHER
H9 cells and samples collected from different time points during endoderm differentiation were dissociated into single cells with TrypLE Express (ThermoFisher Scientific). Zona pellucidae was removed from E4.5 embryos using Acidic Tyrode's solution (Sigma) and washed through three drops of M2 media. Embryos were washed in PBSI (PBS without calcium or magnesium supplemented with 1x Protease Inhibitor Cocktail [PIC], 0.05 U/μL SuperaseIn RNase Inhibitor, 0.1 U/μL Protector RNAse Inhibitor and 0.2% BSA) and dissociated into single cells with TrypLE Express. Nuclei were prepared with Nuclei Extraction buffer and fixed with 0.2% formaldehyde at room temperature for 5 min. Concanavalin A beads were added to the samples to bind nuclei. Preincubated antibody and proteinA-Tn5-MEBdU were added to the nuclei and incubated at RT for 1 hour or overnight at 4°C. Nuclei were washed three times and tagmented at 37°C for 1 hour in the presence of MgCl2. Reaction was stopped with EDTA. Tagmentation was repeated for sequential tagmentation. Followed, reverse transcription was crried out to capture polyA-mRNA transcripts. Three rounds of split-pool barcoding was performed to add combinatory barcodes for each single cells. Nuclei was counted and aliquoted into sublibraries. Reverse crosslinking was performed at 55°C for 1 hour in reverse crosslinking buffer with proteinase K and RNase inhibtors. PMSF was used to inactivate proteinase K. DNA and cDNA were separated by C1 Streptavidin beads. For DNA library, tagmented genomic DNA fragments were purified with Ampure XP beads. Eluted DNA was performed adapter switching and amplified. For RNA library, templated switch was performed and preammplified. MEA adapters were added to the RNA library by Tn5-MEA mediated tagmentation. The concentration of libraries was measured with a Qubit. Samples were run on a Bioanalyzer to check the size distribution of library fragments before submited for seuqencing.