GSM8386175: Human PGCLC KLF4-AID clone11 IAA; Homo sapiens; RNA-Seq
RNA-seq: TNAP+ NANOS3-tdTomato+ PGCLCs were lysed in 50 μL RNA extraction buffer. RNA was extracted with the Arcturus PicoPure kit following the manufacturer's instructions.  Cut&Run:  TNAP+ NANOS3-tdTomato+ PGCLCs were washed and bound to 10 μl Concanavalin A-coated magnetic beads. The cell-bound beads were incubated with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor) containing 0.1% digitonin and 1 μg of KLF4 antibody (ab215036, Abcam) for 2 hours at 4°C on a rotator. After two washes, the beads were resuspended in Protein A/G-MNase fusion protein at 70 ng/ml in digitonin–wash buffer and incubated for 1 hour at 4°C. Following two more washes, the beads were resuspended in ice-cold calcium incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.1% digitonin). After 15 minutes, 2x stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM egtazic acid, 0.1% digitonin, 100 μg/ml RNase A, 50 μg/ml glycogen) was added, and the beads were incubated at 37°C for 30 minutes. The liquid was then transferred to a fresh tube and DNA was extracted using phenol–chloroform. RNA-seq: Library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760) following the manufacturer's protocol. Sequencing was performed on an Illumina NextSeq 500.   Cut&Run: Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645S) from Illumina according to the manufacturer's instructions. PCR enrichment of adaptor-ligated DNA was conducted with the KAPA HiFi Real-Time PCR Library Amplification Kit (Roche, KK2702) as per the manufacturer's guidelines. SPRIselect beads (Beckman Coulter, B23317) were utilized for PCR product clean-up and size selection. Libraries were sequenced for 150 cycles in paired-end mode on the NovaSeq platform.