EXPERIMENT_TITLE: GSM6625136: RH30, HiC, rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: RD, SMS-CTR, Rh30 and Rh41 cells were fixed for 10 minutes at room temperature (23 °C) and was spiked in with 20% mouse myoblasts (C2C12) and lysed gently with HiC buffer to release nuclei, permeabilized in 0.5% SDS for 10 minutes at 62 °C, quenched with 10% Triton X-100, and digested with MboI (200 U, 2h at 37 °C) which was then heat inactivated (20 min, 62 °C). Biotin incorporation was done with biotin-14-dATP (Thermo, Cat# 19524-016) and DNA Polymerase I, Large (Klenow) Fragment (NEB, Cat# M0210) for 1 hour at 37 °C.  Then we performed in situ ligation with T4 DNA ligase (4h room temperature). Nuclei were resuspended in TE and sonicated (5 cycles with shearing 'on' time with 30 seconds 'on' 30 seconds off, using the Active Motif Epi-shear probe sonicator, 30% power. The sheared DNA was reverse crosslinked with SDS and ProteinaseK O/N and pulled down with M-280 Streptavidin Dynabeads (Thermo, Cat#11205D), washed and eluted. End-Repair: Resuspend the bead-bound sample from with 34 μl of TE pH 7.4, and use for the subsequent experimental steps. Add the reagent from End-It DNA End-Repair Kit to make the End-Repair reaction mix. Mix well by pipetting and incubate for 1 h @ RT with slow agitation in a Thermomixer. Resuspend to wash once with 500 μl of Tween Wash Buffer and then once with 500 μl of TE pH 7.4, keeping beads on the magnet throughout washes.   A-tailing to 3′ ends of DNA fragments: Resuspend the bead-bound sample from Step 45 with 32 μl of TE pH 7.4 and add the 3-5'Klenow and buffer to make the A-tailing reaction mix. Incubate for 45 min at 37 °C. Resuspend to wash once with 500 μl of Tween Wash Buffer and then once with 500 μl of TE pH 7.4, keeping beads on the magnet throughout washes.   Linker ligation : Resuspend the bead-bound sample from Step 46 with 22 μl of TE pH 7.4 and add the ligase and adapter to make the Adaptor ligation reaction mix. Incubate for a minimum of 1 h at room temperature with gentle rotation. Resuspend to wash once with 500 μl of Tween Wash Buffer and then once with 500 μl of TE pH 7.4, holding beads magnetically.   Amplify SPICE-C sequencing libraries with unique indexes: Resuspend beads from above step in 50 μl of water, and use 5μL for the following amplification steps. Add 1 μl of Multiplexing PCR FWD (forward) Primer and 1 μl of a REV (reverse) Primer M1-12, with unique barcode. Mix well with quick vortex and table-top centrifugation for several seconds. Add 25 μl of Phusion High-Fidelity PCR Master Mix with HF Buffer and pipette to mix. Remove 25 μl of sample and place in a 200-μl PCR strip tube to prepare for amplification. Run the PCR program for on-bead PCR amplification with 25 μl (half of the total material, after thorough mixing). Check library amplicon fragment lengths with aliquots of 2 μl diluted in 10 μl of water and run with E-Gel EX after 10, 12 and 14 cycles. Desired fragment length should be in the range of 300–800 bp.   library prep. Add 25-μl PCR reaction mix to 25 μl of AMPure beads (1:1 ratio to purify sample from unreacted PCR primers). Mix well and let stand 5 min with beads. Put on a magnet for 5 min. Then, remove the supernatant and wash quickly two times with 80% ethanol with gentle pipette aspiration. Let stand to dry on a magnet for 10–15 min. Add 20 μl of ultrapure water. Mix well and incubate for 5 min. Put on a magnet for 3 min.   Remove the 18 μl of purified indexed SPICE-HiC DNA. Indexed SPICE-C DNA can be stored at−20°C until ready for sequencing. (Modified from Gryder et al. Nat. Protoc., 2020)
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with no precision therapy available for affected patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts to understand chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative rhabdomyosarcoma (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the commonest FP-RMS and FN-RMS cell lines, and compared our data with patient derived xenograft (PDX) models. In our studies we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains, and unique patterns of structural variation. This study enables a comprehensive resource for contextualizing gene regulation events in RMS, and high-depth chromatin interactivity maps for identification of functionally critical chromatin domains in this tumor. Overall design: Human rhabdomyosarcoma (RMS) cell lines RD, SMS-CTR, Rh30 and Rh41 cells were spiked in with 20% mouse myoblasts (C2C12) and fixed with Formaldehyde. Mouse chromatin reads were used as reference to normalize the RMS samples. Two independent biological replicates for each cell line were combined informatically to get contact maps with 30 million valid, long-range cis contacts.
SAMPLE_TITLE: RH30, HiC, rep1
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Pool_Member_@attributes_sample_title: RH30, HiC, rep1
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