GSM8447426: mes-silencer-output5_mes-silencer-output5-7; Mus musculus; OTHER
MEFs were inoculated with 10 % FBS (Gibco; Cat 10437028) and 1% penicillin streptomycin P/S (Sigma; cat P0781) in DMEM (Thermo Fisher; Cat 10566024), MEFs are induced in iCD1 medium(Delicell, Cat 820250). iPSCs and mESCs were cultured in N2B27-2i/LIF medium (Neural Basal, F12,1%Glutamax, 1%NEAA, 1%Sodium pyruvate, 0.1mM β-me, 1%B27, 0.5% N2, 0.5% P/s(optional), mLif(1:8000), 3uM CHIR99021(1:4000), 1 uM PD0325901). The culture conditions of all cells were 37 °C, 5 % carbon dioxide. The construction of the random input DNA library involves three main steps: creating insert fragments, preparing the linearized vector, and seamless assembly cloning. First, the genome is fragmented into 300-400 base pair segments using an ultrasonic disruptor. The library construction kit (Roche; KK8514) is used to add homologous arms and Illumina adapters to these fragments. Next, the Ss-STARR-seq vector is digested with AgeI-HF and SalI-HF enzymes (NEB). The fragmented DNA is then recombined with the linearized vector using Gibson's homologous recombination enzyme (NEB). The quality of the library clones is verified through Sanger sequencing. Forty recombination reactions are set up, and the products are purified using a DNA purification kit (ZYMO; d4013). The purified DNA is transformed into competent cells (Thermo Fisher Scientific; c6400) and cultured in 15 liters of LB medium. When the bacterial culture reaches an optical density (OD) between 1.0 and 1.2, PCR amplification is performed using 100 ng of plasmid as a template. The PCR conditions include initial denaturation at 98°C for 45 seconds, followed by 20 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The amplified products are recovered through 2% agarose gel electrophoresis and prepared for high-throughput sequencing. Cells transfected with the input library were harvested for RNA extraction using TURBOTM DNase (Thermo Fisher Scientific) and Dynabeads Oligo (dT)25 (Thermo Fisher Scientific). The enriched mRNA was reverse transcribed into cDNA using SuperScript TM IV Reverse Transcriptase (Thermo Fisher Scientific) and specific primers. AmbionTM RNase H was then used to degrade any remaining RNA in the cDNA product. The cDNA was purified and amplified via PCR using the HiFi high fidelity enzyme Ready Mix (Roche). The PCR protocol involved initial denaturation at 98°C for 45 seconds, followed by 20 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The PCR products were separated by 2% agarose gel electrophoresis to create the output library for high-throughput sequencing.