GSM8045953: STARR_lib_DNA_input; Mus musculus; OTHER
The candidate enhancer sequences were obtained from PCR of genome DNA (primer sequences in Supplementary Table 3) and cloned between the ORF, a truncated form of GFP, and the polyA site in the hSTARR-seq_ORI vector (Addgene, #99296), which makes each candidate sequence serving as its own barcode. Briefly, the PCR products of each candidate enhancer were purified, pooled together, and ligated with Illumina adaptors in NEBNext Ultr II DNA Library Prep Kit (NEB, E7645S). Subsequently, adaptor-ligated PCR products were cleaned up with AMPure XP beads and amplificated by PCR with library cloning primers (forward: 5`-TAGAGCATGCACCGGACACTCTTTCCCTACACGACGCTCTTCCGATCT, reverse: 5`-GGCCGAATTCGTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) (1 μl adaptor-ligated PCR products, 2.5 μl 10 μM library cloning forward primer, 2.5 μl 10 μM library cloning reverse primer, 25 μl KAPA 2X HiFi HotStart Ready Mix (Roche, KK2602), and 19 μl H2O) with the program of 98 °C for 45 s, (98°C for 15 s, 65°C for 30 s, and 72°C for 45 s) with 5 cycles, and 72 °C for 2 min, which results in that candidate sequences were flanked by overlap sequences with vector around insertion site. The hSTARR-seq_ORI vector was restriction digested by AgeI-HF and SalI-HF and purified for Gibson assembly with ClonExpress II One Step Cloning Kit (Vazyme, C112). The STARR-seq plasmid library was amplified using Illumina i5 and i7 index primers and its quality and complexity were assessed by deep sequencing.