EXPERIMENT_TITLE: GSM7017375: RS411_PCap_HiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Arima promoter capture HiC (Arima: A510008, A303010, A302010) was performed according to the manufacturers provided instructions using unspecified proprietary buffers, solutions, enzymes, and reagents. Briefly, 10 million ALL cells were harvested, suspended in 5ml RT PBS which was brought to 2% formaldehyde by adding 37% methanol-stabilized paraformaldehyde for a 10-minute fixation. The amount of fixed cell suspension equal to 5mg of cell DNA was used for HiC. Cells were lysed with Lysis Buffer and conditioned with Conditioning Solution before their DNA was digested in a cocktail consisting of Buffer A, Enzyme 1, and Enzyme 2. The digested, fixed chromatin was biotinylated using using Buffer B and Enzyme B before being ligated using Buffer C and Enzyme C. The fixed, biotinylated, ligated DNA was then subjected to reversal of crosslinking and digestion of proteins before being purified. 100ul containing 1500ug of purified large proximally ligated DNA was fragmented for 24 cycles (30s on/ 30s off) using a Diagenode Bioruptor Plus bath sonicator. The fragmented DNA was then subjected to two-sided size selection targeting fragments between 200-600bp using AMPure XP DNA purification beads. Size selected DNA was then subjected to biotin enrichment using T1 streptavidin beads. Bead bound, enriched HiC DNA was then subjected to Arima library prep. Briefly, the sample underwent end repair followed by adapter ligation, at which point the sample was then subjected to 10 cycles of PCR amplification. The library DNA was then purified using AMPure XP DNA purification beads. The HiC library was then subjected to Arima promoter capture enrichment. The library was precleared of biotinylated DNA using T1 streptavidin beads before being subjected to promoter enrichment with biotinylated RNA probes. After washing, the captured fragments were then amplified an additional 13 PCR cycles. These libraries were submitted for deep sequencing on an Illumina Nova-seq where >200M 150bp paired-end reads were obtained. Promoter capture Hi-C read length: 150bp
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Although acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer, there is limited understanding of the contribution of inherited genetic variation on inter-individual differences in chemotherapy response, and defining genetic factors impacting therapy failure can help better predict response and identify drug resistance mechanisms. Using inherited noncoding variants associated with chemotherapeutic drug resistance and/or treatment outcome, we mapped these variants to ALL cis-regulatory elements and investigated their gene regulatory potential and genomic connectivity using massively parallel reporter assays and promoter capture Hi-C, respectively. We identified 53 variants with reproducible allele-specific effects on transcription and high-confidence gene targets. Subsequent functional interrogation of the top variant (rs1247117) determined that it disrupted a PU.1 consensus motif and PU.1 binding affinity. Importantly, deletion of the genomic interval containing rs1247117 sensitized ALL cells to vincristine. Together, these data demonstrate that noncoding regulatory variation associated with diverse pharmacological traits harbor significant effects on allele-specific transcriptional activity and impact sensitivity to chemotherapeutic agents in ALL. Overall design: HiC
SAMPLE_TITLE: RS411_PCap_HiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "RS411"}, {"TAG": "cell line", "VALUE": "RS411"}, {"TAG": "cell type", "VALUE": "ALL"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: RS411_PCap_HiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: RS411_PCap_HiC
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