GSM7498138: CT27,EVT, K27ac; Homo sapiens; ChIP-Seq
ChIP was performed on a hTSC line,TSCT-1 (CT27), established in our previous study (Okae et al. Cell Stem Cell 2018) using the ChIP Reagents (NIPPON GENE). The ChIP-seq library was constructed using the Ovation Ultralow System V2 (NuGEN) and sequenced on the Illumina HiSeq 2500 platform (Illumina). The puromycin resistant gene (PuroR) and Cas9 were PCR amplified from the pGIPZ vector (Open Biosystems) and Alt-R S.p. Cas9 Expression Plasmid (IDT), respectively. PuroR, a T2A element, and Cas9 were cloned into the CS-CA-MCS plasmid (kindly provided by H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) using the In-Fusion HD Cloning kit (Takara). The resulting vector was designated as pCS-CA-PuroR-T2A-Cas9. The kanamycin/neomycin resistance gene and the pUC origin were amplified from pCAG-HIVgp (kindly provided by H. Miyoshi), and the human U6 (hU6) promoter was amplified from the pCS-hU6 vector2. The kanamycin/neomycin resistance gene, the pUC origin, the hU6 promoter, 3xFLAG, and a loxP sequence were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). An sgRNA targeting the last exon of SNAI1 (target sequence: 5′-CAC CAA GAG TCC GGC TGC TC-3′), a loxP sequence, and a gRNA target sequence (5′-CCC GAG CAG CCG GAC TCT TGG TG-3′; the PAM sequence is underlined), were inserted between the hU6 promoter and 3xFLAG to generate pU6-sgSNAI1-loxP-FLAG-loxP. The Cre recombinase gene was amplified from the Cre Vector (Santa Cruz) and cloned into the pCS-3G vector2 to generate pCS-3G-Cre. Sequences of the primers used for the vector construction are shown in Supplemental Table. pCS-CA-PuroR-T2A-Cas9 was cotransfected with pCMV-VSV-G-RSV-Rev and pCAG-HIVgp (kindly provided by H. Miyoshi) into 293T cells and lentivirus expressing PuroR-T2A-Cas9 was prepared. The lentivirus was transduced into TSCT-1 cells, and Cas9-expressing cells were selected by 2 mg/ml puromycin for two days. The pU6-sgSNAI1-loxP-FLAG-loxP vector was transfected into the Cas9-expressing TSCT-1 cells using an electroporator (CUY21Vitro-EX; BEX) with the following setting: Poration pulse [125 V, 10 ms]; Driving pulse [20 V, 50 ms, 5 pulses]. After 200 mg/ml G418 selection for 5 days, lentivirus expressing Cre was transduced into the cells to delete the kanamycin/neomycin resistant gene, the hU6 promoter, and the sgRNA sequence. Then, single-cell cloning was performed, and clones with an in-frame insertion of 3xFLAG were identified by Sanger sequencing. This knock-in strategy is based on homology-independent targeted integration3. One of the obtained clones had a homozygous in-frame insertion of 3xFLAG (Supplemental Figure) and was used for ChIP-seq. ChIP was performed on the clone with 3xFLAG-tagged SNAI1 using the ChIP Reagents (NIPPON GENE) and an anti-FLAG antibody (Sigma, Clone #M2). Immunoprecipitation was carried out in IP buffer containing 400 mM NaCl instead of 150 mM to reduce the background signal. The sequencing adapters were a mixture of two adapters (Adapter-T and Adapter-C) in equal amounts4. Adapter-T with a 3'-T overhang was prepared by annealing (5'-CTA CAC GAC GCT CTT CCG ATC TT-3') and (5'-AGA TCG GAA GAG CAC ACG TCT GAA-3'; 5' phosphorylated). Adapter-C with a 3'-C overhang was prepared by annealing (5'- CTA CAC GAC GCT CTT CCG ATC TC-3') and (5'-AGA TCG GAA GAG CAC ACG TCT GAA-3'; 5' phosphorylated). Adapter-C was added to increase the ligation efficiency. The ChIP-seq library was constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and sequenced on the Illumina HiSeq 2500 platform (Illumina).