title: GSM3902827: B250C11: TC71_WT_CTCF_rep1; Homo sapiens; OTHER
library_construction_protocol: Several adaptations were made and are detailed below in an overview of the otherwise original protocol described by Mumbach and colleagues (PMID: 27643841). Cells were plated 2 days before starting the experiment to reach a 90-95% confluence at the time of fixation (1% of methanol-free formaldehyde (28908, Thermo-Scientific) in freshly prepared respective cell culture media, 20ml/T150 flask). Cells were fixed at room temperature for 10 min under gentle shaking platform (50mvt/min). 2 mL of glycine solution (2M) were added and cells were incubated for 5 additional min at room temperature under gentle shaking platform. The supernatant was removed and cells were washed 3 times with PBS at room temperature. Cells were scraped vigorously with ice cold PBS supplemented with protease inhibitor cocktail tablets (11836145001, Roche), and flushed five time through a syringe with a 21 gauge needle (301155, BD Microlance). In situ contact libraries were performed starting from 15 million nuclei digested overnight at 37°C with Mbo1 (R0147M, New England Biolabs). After proximity ligation (4 h at room temperature), the nuclear pellet was sonicated and the chromatin immunoprecipitation step was performed using the iDeal ChIP-seq kit for transcription factors (Diagenode) according to the supplier's recommendation with some modification. Nuclei were resuspended in IL1b and IL2 buffer following Diagenode protocol and all centrifugations were performed at 4°C for 5 min at 1,950 RCF for these steps. The chromatin was sonicated (5 million nuclei per tube) with Bioruptor pico (Diagenode) for 10 cycles (30-sec on, 30-sec off) set at position “high”. The tubes were pooled and the chromatin was clarified by centrifugation at 4°C for 10 min at 16,000 RCF. Sonicated chromatin from 11 and 3 million nuclei were used respectively for CTCF and H3K27ac immunoprecipitation step using the equivalent of 3 ChIP reactions pooled in one tube for each HiChIP reaction (final volume was 1050 µl/tube). Immunoprecipitation was then carried out following Diagenode kit instructions by multiplying all reagents by a factor 3 until end of elution step (50µl). Biotin capture was performed following Mumbach et al. protocol (PMID: 27643841). Library for Illumina sequencing were prepared using 10 ng of chromatin and 0.5 µL of TN5 (15028211, Illumina). A first PCR in a final volume of 50µl with 5 cycles was performed (72 °C for 5 min, 98 °C for 1 min, then 5 cycles at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min). To determine how many additional PCR cycles were required for optimal library preparation, a QPCR with: 5µL of the first PCR product, 1 μL of Nextera Ad1_noMX and Nextera Ad2.X (each at 1.25 μM), 5 µl of phusion HF 2X (M0531S, New England Biolabs), 0,75 µL Evagreen® 20X (31000, Biotium) and 2,25 µL of water was runned (same program as first PCR but with 30 cycles). The optimal number of additional cycle was determined for each library by setting a threshold just before reaching the end of exponential amplification step. The 45µl left from the first PCR were further amplified using additional PCR cycles as determined above. Size selection was performed using Ampure XP beads (A63881, Beckman Coulter) to capture fragments greater than 300 bp. Libraries were quantified and analyzed using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and LabChIP (Perkin Elmer). A first validation of HiChIP experiments was performed using 150 bp paired-end sequencing on MiSeq-microV2-300-PE150 (Illumina). Deep 75 bp paired-end sequencing was then performed on HiSeq 2500 or NovaSeq 6000 systems (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3902827