hTERT1-RPE1, RB WT, S
Homo sapiens
Protocols: Micro-C experiments were performed using the Micro-C kit (10X wash buffer, NWB solution, chromatin capture beads, streptavidin beads, 20% SDS, crosslink reversal buffer, MNase enzyme mix, 10X nuclease digest buffer, 100 mM MgCl2, 0.5 M EGTA, end polishing buffer, end polishing enzyme mix, intra-aggregate ligation buffer, intra-aggregate ligation enzyme mix, bridge, 5X bridge ligation buffer, bridge ligase, proteinase K, 250 mM DTT, and HotStart PCR Ready Mix; Dovetail Genomics, Cat. # 21006) via Dovetail Genomics' Micro-C service (Cat. # 20020). In short, frozen hTERT1-RPE1 cells were resuspended in 1X PBS, crosslinked with crosslinked using 3 mM disuccinimidyl glutarate (DSG; Thermo Fisher Scientific, Cat. # A35392) and 1% formaldehyde. All cells were cultured at 37ºC with 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 25 mM glucose, 2 mM glutamine, 5% fetal bovine albumin, and antibiotics (penicillin, 100 units/mL; streptomycin, 100 μg/mL). CRISPR/Cas9 targeting exon 1 of the RB1 gene was utilized to prepare a RB KO in hTERT1-RPE1 cells. G1 synchronization was performed via contact inhibition through maintaining cells for 6 d in a confluent state. Cells were subsequently released to S phase via trypsinization. RB1 knockdown or the replacement of endogenous RB with the constitutively active RB (i.e., RB∆CDK) were done in cells transduced with pINDUCER11 (Addgene, Cat. # 44363) or pINDUCER20 (Addgene, Cat. # 44012) constructs. Doxycycline induction was performed by treating cells with doxycycline (Sigma-Aldrich, Cat. # D9891) at 0.5 μg/mL concentration. MNase reaction was performed using 0.5 μL of MNase enzyme mix in a 100 μL solution of 1X nuclease digest buffer and was stopped by addition of 5 μL of 0.5 M EGTA. Digested chromatin was solubilized by supplementing with 3 μL of 20% SDS and was captured by chromatin capture beads. End polishing was performed using the end polishing enzyme mix at the end polishing buffer, and crosslinks were reversed by 1X crosslink reversal buffer supplemented with proteinase K. DNA was purified using SPRIselect beads (Beckman Coulter, Cat. # B23317). The sequencing library was prepared using the Dovetail Library Module for Illumina (end repair enzyme mix, end repair enzyme buffer, ligation enhancer, ligation enzyme mix, adaptor for Illumina, and USER enzyme mix; Dovetail Genomics, Cat. # DG-LIB-001). End repair enzyme mix and end repair buffer supplemented with 12 mM DTT were utilized for end repair, which were followed by adapter ligation and USER digest. 2.5 μL adaptor for Illumina, 30 μL ligation enzyme mix, and 1 μL ligation enhancer were used for the 33.5 μL reaction mixture. DNA was then purified with SPRIselect beads (Beckman Coulter, Cat. # B23317), ligation captured via streptavidin beads, and underwent Index PCR using the HotStart PCR ready mix. Size selection was performed using SPRIselect beads (Beckman Coulter, Cat. # B23317).
disease
normal
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
retina
developmental stage
adult
genotype
RB WT
isolate
not applicable
growth condition
S synchronization via trypsinization
cell_line
HTERT1 RPE-1
cell_type
retinal pigment epithelium
geographic location (country and/or sea)
not collected
ENA-LAST-UPDATE
2023-12-04