title: Illumina HiSeq 2500 sequencing; Functional analyses of human LUC7-like proteins involved in splicing regulation and myeloid neoplasms
library_construction_protocol: Experiments were performed using the single-end crosslinking immunoprecipitation (seCLIP) protocol [Van Nostrand (seCLIP) et al., 2017]. K562 cells with CRISPR-epitope tagged LUC7L, LUC7L2 and LUC7L3 were transferred to 10cm plates for crosslinking using a Stratalinker 2400 with 254 nm light at 400 mJ/cm2. Non-crosslinked controls were placed in PBS but not irradiated. Samples were sonicated using the Biorupter (Diagenode) on the \"low\" setting for 30 second intervals followed by 30 second pauses for 5 minutes at 4°C. DNase I (2 μl, Invitrogen AM2239) and RNase I (10 μl, 1:25 RNase I:PBS solution, Ambion AM2295) were added to the sample and mixed in the Thermomixer (1200 rpm, 37°C, 5 minutes). LUC7-like protein-RNA complexes were immunoprecipitated with V5 antibody (anti-V5, Bethyl, A190-120A) followed by stringent washes using high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, in RNase/DNase free H2O) and wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20, in RNase/DNase free H2O). Following washing, RNA was dephosphorylated using FastAP and T4 PNK and a 3' RNA adapter was ligated onto the RNA. Samples were denatured and resolved using SDS-PAGE on a 4-12% Bis-Tris gel. The gel was transferred to PVDF membrane in NuPAGE transfer buffer and the region on the membrane corresponding to the respective LUC7-like proteins and 75 kDa above were isolated for RNA extraction. The isolated RNA was reverse transcribed (AffinityScript Kit 600107) followed by the addition of a 5' linker to the cDNA. The final libraries were amplified using Q5 PCR mix (NEB) and the 175-350bp region corresponding to the library was excised from a 3% low-melting temp agarose gel. The libraries were quantified on the BioAnalyzer D1000 then sequenced on the Illumina HiSeq2500. K562 cells were grown in RPMI, 10%  FBS, and 100 U/mL penicillin-streptomycin. Experiments were performed using the single-end crosslinking immunoprecipitation (seCLIP) protocol [Van Nostrand (seCLIP) et al., 2017]. K562 cells with CRISPR-epitope tagged LUC7L, LUC7L2 and LUC7L3 were transferred to 10cm plates for crosslinking using a Stratalinker 2400 with 254 nm light at 400 mJ/cm2. Non-crosslinked controls were placed in PBS but not irradiated. Samples were sonicated using the Biorupter (Diagenode) on the \"low\" setting for 30 second intervals followed by 30 second pauses for 5 minutes at 4°C. DNase I (2 μl, Invitrogen AM2239) and RNase I (10 μl, 1:25 RNase I:PBS solution, Ambion AM2295) were added to the sample and mixed in the Thermomixer (1200 rpm, 37°C, 5 minutes). LUC7-like protein-RNA complexes were immunoprecipitated with V5 antibody (anti-V5, Bethyl, A190-120A) followed by stringent washes using high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, in RNase/DNase free H2O) and wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20, in RNase/DNase free H2O). Following washing, RNA was dephosphorylated using FastAP and T4 PNK and a 3' RNA adapter was ligated onto the RNA. Samples were denatured and resolved using SDS-PAGE on a 4-12% Bis-Tris gel. The gel was transferred to PVDF membrane in NuPAGE transfer buffer and the region on the membrane corresponding to the respective LUC7-like proteins and 75 kDa above were isolated for RNA extraction. The isolated RNA was reverse transcribed (AffinityScript Kit 600107) followed by the addition of a 5' linker to the cDNA. The final libraries were amplified using Q5 PCR mix (NEB) and the 175-350bp region corresponding to the library was excised from a 3% low-melting temp agarose gel. The libraries were quantified on the BioAnalyzer D1000 then sequenced on the Illumina HiSeq2500.