title: GSM8760417: H9-siNIPBL-N1_ChIP_INPUT_Brep1_Trep1; Homo sapiens; ChIP-Seq
library_name: GSM8760417
library_construction_protocol: RNA-seq: Total RNA was extracted from control (Ctrl) and siRNA-treated H9 cells, as well as from their in vitro differentiated stages, using Trizol reagent (Invitrogen). Ribosomal RNA was subsequently removed with the Ribo Minus Transcriptome Isolation Kit (Invitrogen, K1550). RNA concentrations were quantified using the Qubit RNA HS Assay Kit (Thermo Fisher) and fragmented randomly by adding fragmentation buffer. cDNA synthesis was performed with the RNA template and random hexamer primers. Following terminal repair, A ligation, and the ligation of sequencing adapters, the double-stranded cDNA library was generated through size selection and PCR enrichment. Two independent biological replicates for each sample were then sequenced using paired-end 50 bp reads on an Illumina NovaSeq 6000 system. In-situ Hi-C: In-situ Hi-C libraries were generated using the DpnII restriction enzyme, following previously established protocols. In summary, 2.5 million cells from each differentiation stage were crosslinked using 1% formaldehyde, quenched with glycine, washed with PBS, and permeabilized to isolate intact nuclei. The nuclear DNA was then digested with DpnII, with 5'-overhangs filled in with biotinylated dCTPs and dA/dT/dGTPs to create blunt-end fragments. These fragments were subsequently ligated, reverse-crosslinked, and purified using standard DNA ethanol precipitation methods. The purified DNA was sonicated to generate fragments of 200-500 bp in size and captured using streptavidin beads. The standard Illumina TruSeq library preparation protocol was followed, which included end-repair, A-tailing, and ligation with universal adapters, with the beads being washed twice in Tween Washing Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) between each step. The DNA attached to the beads was PCR amplified using barcoded primers and KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) for 5 to 12 cycles to ensure adequate DNA quantity for sequencing. The libraries were then paired-end sequenced on an Illumina NovaSeq 6000 platform. More than two biological replicates were created, and the replicates were combined for all analyses after confirming a high level of correlation. HiChIP: HiChIP libraries were generated using control (ctrl) and siRNA-treated H9 cells, along with their differentiated counterparts at various stages, as previously described, with some modifications. In brief, cells were collected and crosslinked with 1% formaldehyde for 10 minutes at room temperature, then quenched with glycine, washed with PBS, and stored frozen at -80°C. For library preparation, the fixed cells were gently homogenized using pestle A in Hi-C lysis buffer to release the nuclei, followed by digestion with DpnII, biotin-dCTP fill-in, and re-ligation. After ligation, chromatin was sheared by sonication into fragments of 200–500 bp and pre-cleared with Protein A and G Dynabeads at 4°C for 2 hours. Then, it was precipitated using Protein A or G Dynabeads that had been pre-incubated overnight with the appropriate antibodies to enrich the ligation products associated with specific proteins. Tagmentation of the immunoprecipitated chromatin was performed using a Tn5 transposase mixture on the beads. Following elution, reverse crosslinking, and ethanol precipitation, a second pull-down with streptavidin beads was conducted to enrich biotin-labeled ligation products. Finally, on-bead PCR amplification was performed to prepare the libraries for Illumina sequencing. Two biological replicates were created for each sample. ChIP-seq: ChIP-seq experiments were conducted as previously described. Following the removal of the medium, cells were crosslinked in 1% formaldehyde in PBS at room temperature for 10 minutes and then quenched with glycine. The cell pellets, rinsed with PBS, were flash-frozen in liquid nitrogen and stored at -80°C or used immediately. After lysing the cells, chromatin was sonicated to generate fragments ranging from 200 to 500 bp, pre-cleared using Protein A or G Dynabeads at 4°C for 2 hours, and then precipitated overnight with antibodies at 4°C. The immunoprecipitated chromatin was then treated with a Tn5 transposase mixture on the beads, after which it was eluted, reverse crosslinked, and purified using standard techniques. The purified DNA was amplified with Illumina Nextera barcoded primers using KAPA SYBR FAST qPCR Master Mix, undergoing 5 to 12 PCR cycles to ensure sufficient DNA for sequencing. ATAC-seq: Control (ctrl) and siRNA-treated H9 cells, along with their in vitro differentiated derivatives, were collected for ATAC-seq analysis. For the SC-β organoids, an electric homogenizer was used to break them down into small clusters of cells within 10 seconds. The cells were washed with PBS, and the nuclear membranes were disrupted by immersing them in lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) on ice. The nuclei were then washed once with cold lysis buffer devoid of NP40 and digitonin before being incubated in a Tn5 transposase mixture (25 µl of 2x TD buffer, 2.5 µl Tn5 (final concentration of 100 nM), 16.5 µl PBS, 0.5 µl of 1% digitonin, 0.5 µl of 10% Tween-20, and 5 µl of H2O) at 37°C for 20 minutes, with occasional shaking. After the reaction was complete, DNA was extracted using the MinElute Kit (Qiagen). The purified, tagmented DNA was utilized to construct Illumina Nextera libraries, which were subsequently sequenced on a NovaSeq 6000 instrument. Truseq libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), A-tailed by adding adenosine to the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligated at room temperature for 1 hr with T4 DNA ligase (New England Biolabs). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix. Nextera libraries were amplified with Nextera barcoded primers using the KAPA SYBR FAST qPCR Master Mix. Illumina libraries were sequenced paired-end at 50 bp length on Illumina Hiseq2500 v4 or NovaSeq 6000 instruments.