GSM5210221: SOX15_cuttag rep3; Homo sapiens; ChIP-Seq
The hESCs, 4i cells and EBs were washed once in PBS one time and dissociated into single cells with TrypLE Express enzyme or Trypsin. Then the dissociated cells were collected for next experiments. In order to study the distribution of SOX15 in hPGCLC, we used NovoNGS ® CUT&Tag 2.0 High-Sensitivity Kit (N259-YH01, Novoprotein) to capture SOX15-binding sites. The experimental process was performed according to the manufacturer's instructions. In brief, 1×105 day 4 SOX15-3×Flag-p2A-EGFP hPGCLC were prepared and immobilized on concanavalin A beads. Beads are incubated with a Flag primary antibody (F1804, Sigma), followed by incubation with a secondary antibody anti-Mouse IgG (ab6708, Abcam). Beads were washed and incubated with pA-Tn5. Tn5 was activated by addition of Mg2+ and incubated at 37°C for 1 h. Reactions were stopped by the addition of 10 µL 0.5M EDTA, 3 µL 10% SDS and 2.5 µL 20 mg/mL Proteinase K to each sample. DNA was extracted with phenol-chloroform and constructed CUT&Tag library according to the manufacturer's instructions. Library was quantified by Equalbit dsDNA HS Assay Kit (Vazyme, EQ111-01) using Qubit™ 4 Fluorometer (Invitrogen, Q33226). Libraries were subjected to paired-end 150 bp sequencing on NovaSeq platform at Novogene.