title: GSM8398133: eCLAP_KRBP72_mito_genome_rep3; Trypanosoma brucei brucei; OTHER
library_construction_protocol: KRBP72-MHT tagged PCF cells were grown to ~1x107 cells/mL, and 3x109 cells were collected per experiment and three biological experiments were performed. Cells were pelleted at 5000g for 10 min, washed with ice-cold PBS containing 6 mM sucrose, and resuspended in 32 mL of the same buffer and centrifuged again. Cells were resuspended in in 50 mL SM media without FBS, distributed into two prechilled 10 cm Petri dishes, and crosslinked by irradiation at 800 mJ/cm2  twice using a Stratalinker Crosslinker. Post-crosslinking, cells were pelleted, washed with PBS, snap-frozen in liquid nitrogen, and stored at −80°C. Crosslinked cells were centrifuged at 6000g for 10 min at 4°C, resuspended in DTE buffer (1 mM Tris (pH 8), 1 mM EDTA, Complete protease inhibitor cocktail, RNaseOUT), homogenized on ice, and mixed with 60% sucrose before centrifuging at 15,800g for 10 min. The pellet was resuspended in  7.5 mL STM buffer (250 mM sucrose , 20 mM Tris (pH 8), 2 mM MgCl2, 0.2 mM CaCl2) with DNase I and incubated on ice for 1 hour, followed by the addition of 7.5 mL STE buffer (250 mM sucrose, 20 mM Tris (pH 8), 10 mM EDTA) and centrifugation at 13,000 rpm. The pellet was flash-frozen in liquid nitrogen and stored at -80°C. For lysis, the pellet was resuspended in lysis buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.5% NP-40, 1% Triton X-100, Complete protease inhibitor cocktail, RNase Inhibitor), rotated at room temperature for 20 min, centrifuged at 13,000 rpm, and the supernatant was collected. For RNase treatment, reactions in 1 mL volume included DNase TURBO, 1M CaCl2, and RNase I at specified dilutions, incubated at 37°C in a thermomixer at 1500 rpm for 5 min, followed by termination with EDTA and RNase inhibitor. For immunoprecipitation, nickel-NTA agarose beads (Invitrogen) were equilibrated in binding buffer, added to the lysate, rocked at 4°C for 2 hours, washed with N150 buffer, and incubated with denaturation buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.5% NP-40, 8M Urea with Complete protease inhibitor cocktail). Beads were washed, then resuspended in CIAP buffer with CIAP for 20 min at 37°C, washed with PNK buffer, and resuspended in PNK buffer with 32P-[gama-ATP] and T4 PNK for 30 min at 37°C. After adding 1 mM ATP and incubating, beads were washed, resuspended in SDS loading buffer, boiled, and the supernatant was loaded onto an SDS-PAGE gel for Western blotting, with the membrane exposed to a phosphor screen before antibody blotting against the myc tag. eCLIP libraries were prepared according to the RBP-eCLIP kit v1.01R (Eclipse Bioinnovations) protocol with modifications. Specifically, 100 µL of His-tag isolation Dynabeads® (Thermo Fisher Scientific) were employed for immunoprecipitation, bypassing the need for magnetic beads conjugated to antibodies. The protocol was further streamlined by omitting size-matched input processing and gel purification post-AMPure PCR cleanup. Library quality was assessed using an Agilent Bioanalyzer,followed by single-end sequencing (75 cycles) on an Illumina NextSeq 550 v2.5 (High Output)at the CWRU Genomics Core.