title: GSM8910759: RNA-seq shGFP biol rep2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-seq: Snap-frozen RNA samples were extracted using Mei5bio M5 HiPer Total RNA Extraction Reagent (MF034-01) including deoxyribonuclease digestion. CUT&Tag: Concanavalin A-coated magnetic beads (Novoprotein, N253-YH01) were introduced to the resuspended cells and incubated at room temperature to facilitate the binding of nuclei. The nonionic detergent digitonin was utilized to permeabilize the cell membrane. Then, ΔNp63α antibody (Cell Signaling Technology, 67825 S), H3K27ac antibody (Cell Signaling Technology, 4353), the secondary antibody and Hyperactive pA-Tn5 Transposase(The pA-Tn5 used in this study was kindly provided by Prof. Zhonghan Li, Sichuan University) were incubated with the cells that had been bound by ConA beads. Consequently, the highly active pA-Tn5 transposase can precisely cleave the DNA fragments that were bound with the target protein. Next, the cells were placed in fragmentation buffer and incubated at 37°C for one hour. Additionally, the cleaved DNA fragments can be ligated to P5 and P7 adaptors using Tn5 transposase. RNA-seq: The reverse  transcription of RNA to cDNA and the construction of the sequencing library were carried out by Novogene. RNA-seq was performed at Novogene. CUT&Tag: The resulting libraries are then amplified through PCR with the P5 and P7 primers. The DNA fragments were then extracted, purified, and used to construct libraries with the kit's reagents. CUT&Tag sequencing was performed at Novogene, China using the Illumina HiSeq 2000 platform to generate approximately 12 Gb of 150 bp paired-end reads per sample.