GSM7438840
Chromatin was extracted and fixed as described previously (Downes et al., 2022), following the protocol for low input and adapting centrifugation steps to organoid cells (400 g). Briefly, 25,000 organoid cells were trypsinized, collected in 1ml medium and crosslinked with 134µl of 16% formaldehyde (1.89% final; ThermoFisher Scientific, #10751395) while rotating for 10 min at room temperature. Cells were quenched with 162µl 1M cold glycine, washed with cold PBS and lysed for 20 minutes at 4°C in 500µl lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40, supplemented with cOmplete proteinase inhibitors (Roche)) prior to snap freezing on dry ice. Fixed chromatin was stored at -80°C. Capture-C libraries were prepared as described previously, following the recommendations for Low Input samples (Downes et al., 2022). Briefly, lysates were thawed on ice, pelleted and resuspended in 200µl 1x DpnII buffer (New England Biology). The nuclei were subsequently permeabilized with SDS (ThermoFischer Scientific, AM9820, 0.28% final concentration, 1hr, 37°C, interval shaking 500rpm, 30 sec on/off), quenched with Triton X 100 (1.67% final concentration, 1hr, 37°C, interval shaking 500rpm, 30 sec on/off) and digested for 24 hours with 3x10µl DpnII (homemade, 37°C, interval shaking 500rpm, 30 sec on/off). The digested chromatin was then in situ ligated with 8µl T4 Ligase (240U, ThemoFirsher Scientific, EL0013) in a volume of 1440µl (20 hours, 16°C). Following this, the nuclei containing ligated chromatin were pelleted to remove any non-nuclear chromatin, reverse-crosslinked and the ligated DNA was phenol-chloroform purified. The sample was resuspended in 100µl water and sonicated 13x (Bioruptor Pico, 30sec on/off) to obtain fragments of ~200bp, followed by a concentration using AmpureX beads (Beckman Coulter #A63881, ratio: 1.8x). All material was adaptor-ligated and indexed in one reaction using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs: E7645L) and NEBNext Multiplex Oligos for Illumina Primer sets 1 (New England Biolabs #E7335S/L) and 2 (New England Biolabs #E7500S/L). The libraries were amplified with 8 PCR cycles using Herculase II Fusion Polymerase kit (Agilent #600677) and their appropriate size assessed via Bioanalyzer. Libraries were next hybridized in the following way: For each promoter-containing DpnII restriction fragment we designed one-two capture probes (aligned to mm10, 70bp each) using the online tool designed by the Hughes lab (CapSequm2: http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi) with the following filtering parameters: Duplicates: <2, Density <30, SRepeatLength <30, Duplication: FALSE. Note that only one capture probe could be designed for the first Ciita TSS (Supplementary Table 6). The 5' biotinylated probes were pooled at a 2.9 nM each and the samples were multiplexed by mass prior to hybridization (2ug each, according to Qubit dsDNA BR Assay, Invitrogen #Q32850). Hybridization was carried out using the KAPA HyperCapture system (Roche, KAPA HyperCapture Reagent Kit #9075828001, KAPA HyperCapture Bead Kit #9075798001) according to Roche protocol for 72 hours followed by a 24 hours hybridization (double Capture). During the PCR amplification steps libraries were amplified for 18 cycles to ensure all of the low input sample is captured. Captured libraries were quantified by qPCR using the KAPA Sybr Fast Universal kit (KAPA #KK4602) and sequenced on Illumina NextSeq 500.