title: Mpp8 regulates the activity of LIF/STAT3 pathway in mouse embryonic stem cells
design: Mpp8 regulates the activity of LIF/STAT3 pathway in mouse embryonic stem cells
library_construction_protocol: Total RNA was purified by RNeasy Minikit (Qiagen) according to the manufacturer's manual. RNA concentration was determined by Nanodrop, and 500 ng of total RNA was used for library construction using KAPA Stranded mRNA-Seq Kits. Sequencing was performed on Illumina HiSeq 2500 machines with 125 bp pair-end mode. ESCs were cultured in a medium comprising of DMEM (Procell, WH0021U211) supplemented with 10% fetal bovine serum (Labtech, FCS-SA/500), 1% L-Glutamine (Gibco, 25030-081), 1% Penicillin-Streptomycin (Gibco, 15140-122), 0.1% β-Mercaptoethanol (Sigma, M3148), and 103 U/ml mLIF (Genescript, Z03077). For differentiation, ESCs were dissociated using 0.05% Trypsin-EDTA (Gibco, 2072820) and resuspended in ES medium without LIF. Approximately 6~8×105 ESCs were then plated in low-attachment petri dishes and incubated at 37°C with 5% CO2 to induce the formation of embryoid bodies (EBs). To obtain homogeneous AID-tagged Mpp8 ES cells, Rosa26-OsTIR1 ESCs were transfected with the linearized PL-Mpp8-Flag-AID-EGFP vector, the corresponding sgRNA vector, and Cas9-encoding vector using Lipo8000 transfection reagent (Beyotime, C0533FT). Following transfection, colonies were selected using a combination of 250 μg/ml G418 (Sangon Biotech, GDJ958) and 1 μg/ml of Puromycin (Sigma, P8833). Finally, clones were selected for genotyping. To generate inducible Mpp8 overexpressing ESCs, the Mpp8 cDNA was amplified and inserted into the pPBH-TREtight-MLC-EGFP vector. The Mpp8 overexpression vector and PBase vector were then transfected into Mpp8AID ESCs. Colonies were selected for genotyping after being cultured in the selection medium containing 100 μg/ml hygromycin (Invitrogen, 10687010). The culture medium supplemented with 1 μg/ml Doxycycline (Sigma, 3219-99-6) was used to induce the overexpression of Mpp8. Total RNA was extracted using the RNeasy Minikit (Qiagen) based on the manufacturer's instructions. Library was prepared using KAPA Stranded mRNA-Seq Kits based on the manufacturer's instructions.