title: GSM4220246: GSK126_1umol_E12.5plus2d_Epcam_1_10; Mus musculus; RNA-Seq
library_construction_protocol: For scRNA-seq, livers were dissociated into single cells by treatment with 0.25% trypsin at 37°C. The reaction was terminated by the addition of 0.4 volumes of FBS. The single-cell suspensions were centrifuged at 100 × g for 5 min at 4 °C, and the resulting pellets were re-suspended with PBS containing 1% FBS. Cells were incubated with the DLK-FITC antibody (MBL, D187-4, 1:100) and/or EpCAM-APC antibody (eBioscience, 17-5791-82, 1:50) at 4 °C for 20 min. After washing, cells were sorted with a BD FACS Aria SORP cell sorter (BD Bioscience). Dead cells were excluded by DAPI (Sigma, D9564, 0.5 µg/mL). The generation of cDNA were performed according to Smart-seq2 method. For scRNA-seq, two nanograms of cDNA was used to prepare libraries using a TruePrep DNA Library Prep Kit (Vazyme, TD502). Four-cycle PCR using biotinylated index primers introduced a biotin tag to the 3' end of the cDNA. After cDNA fragmentation using a Bioruptor Plus sonicator (Diagenode), the 3' terminals of the cDNA were captured using Dynabeads MyOne Streptavidin C1 (Thermo Fisher, 65002). Thirty nanograms of cDNA was used to construct libraries using a Kapa Hyper Prep Kit (Kapa Biosystems, KK8505). For N-ChIP and CUT&RUN ChIP, we collected approximately 20,000 cells for each ChIP-seq, and used antibodies against H3K4me3 (Abcam, ab8580, 1ng), H3K27me3 (Millipore, 07-449, 1ng), H3K4me1 (Millipore, 07-436, 1ng), H3K27ac (Active motif, 39133, 1ng) and EZH2 (Cell Signaling Technology, 5246, 1:100). N-ChIP was carried out following the protocol of Brind'Amour et al. (2015), and CUT&RUN ChIP-seq was performed following the protocol of Peter J Skene et al. (2018). For N-ChIP and CUT&RUN ChIP, libraries of ChIP DNA were constructed with the Ultra DNA Library Prep Kit for Illumina (New England Biolabs, E7370) or the KAPA Hyper Prep Kit (Kapa Biosystems, KK8502). DNA purification was conducted with VAHTS DNA Clean Beads XP beads (Vazyme, N411-03). The quality of the libraries was assessed with an Advanced Analytical Fragment Analyzer (AATI). For bulk cell RNA-seq, livers were dissociated into single cells by treatment with 0.25% trypsin at 37°C. The reaction was terminated by the addition of 0.4 volumes of FBS. The single-cell suspensions were centrifuged at 100 × g for 5 min at 4 °C, and the resulting pellets were re-suspended with PBS containing 1% FBS. Cells were incubated with the DLK-FITC antibody (MBL, D187-4, 1:100) at 4 °C for 20 min. After washing, cells were sorted with a BD FACS Aria SORP cell sorter (BD Bioscience). Dead cells were excluded by DAPI (Sigma, D9564, 0.5 µg/mL). Total RNA was extracted from approximately 50,000 cells using the RNeasy Micro Kit (Qiagen, 74004). For bulk cell RNA-seq, libraries were constructed with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, #E7490) and the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs, #E7420).
identifiers.namespace: GEO
identifiers.id: GSM4220246