{"database": "metadata", "table": "run_metadata", "rows": [[10210, "ERR6474216", "ERX6101492", "ERS7377022", "ERP131171", "PRJEB46937", "RNA Affinity Purification followed by sequencing RAP seq allows identification of transcriptome wide RBP binding sites using recombinant RBPs and total purified cellular RNA", "E-MTAB-10834", "Other", "RAP seq is a new method that provides in vitro derived RNA Interactomes for any given RBP. In RAP seq a recombinant RBP is produced as fusion with a HaloTag which is used to recover and purify the RBP of interest. The RBP Halo fusion is then incubated with fragmented total RNA derived from any given sample of interest. The bound RNA fragments are subsequently eluted and cloned using a small RNA library preparation protocol for sequencing the pool of bound molecules on an Illumina NGS platform.   In this study  RAP seq was used to identify RNA Interactomes of 26 novel RBPs aka non canonical RBPs newly discovered in proteome wide studies as RNA binders. RAP seq was also used to profile vertebrate HuR orthologs and described the biochemical evolutionary differences and similarities of the 6 orthologs profiled. Cancer associated IGF2BP1  IGF2BP2 and IGF2BP3 variants were also profiled and transcriptome wide changes in their RNA Interactomes with respect to the wild type IGF2BPs were reported. Also a transcriptome wide cooperative binding assay was perfomed to evaluate the cooperative roles of HuR and PTBP1 in binding to their native RNA targets.  In addition  a typical RAP seq substrate fragmented total RNA if reverse transcribed into cDNA and than in vitro transcribed again using a T7 RNA Polymerase can be depleted of any native endogenous RNA modifications and RAP seq assyas perfomed in parallel with the native substrate and the T7 RNAP produced one allowed us to discern the m6A dependency in transcriptome wide binding events for YTHDF1  hence with RAP seq we also report T7 RAP seq.", "ENA FIRST PUBLIC:2023 08 11|ENA LAST UPDATE:2023 08 11", null, "Protocols: HepG2 were obtained from American Type Culture Collection ATCC  Rockville  MD. The cell line was mycoplasma free when periodically tested with Mycoplasmacheck Eurofins Genomics. HepG2 were cultured in T75 flasks at 37 \u00b0C and 5 % CO2 atmosphere using Dulbecco's Modified Eagle Medium DMEM supplemented with 1/100 Penicillin/Streptomycin P/S  Sigma and 10 % fetal bovine serum Hyclone  GE healthcare. HepG2 cells were maintained by splitting 1/6 three times a week  done by aspirating the medium  gently washing the cells with phosphate buffered saline PBS  without xxx+  Sigma and detaching them with 2 mL of a trypsin EDTA solution 0.05%  Sigma for 3 5 min. Trypsin EDTA was inactivated with a minimum of 10 fold surplus of culture medium before a cell fraction was passaged Substrate preparation: DNAse treated and column cleaned total RNA was fragmeted at 95 degrees celsius in Tris HCl 80 mM and MgCl2 8 mM final concentrations for 4 minutes and 30 seconds. post fragmentation  the RNA was cleaned using 1 volume of RNA CleanXP Ampure beads and 1 volume of isopropanol; post 2 80% Ethanol washes the RNA was eluted in half the volume of AMPure beads used. three prime dephosphorylation was then performed with T4PNK in TAM buffer Tris Acetate 50 mM  Magnesium Chloride 10 mM  pH8.5 at room temperature for 4 hours followed by AMPure clean up and subsequently five prime phosphorylated with T4PNK in T4PNK buffer with addition of ATP at 37 degreees celsius for 1 hour according to NEB manufacturer instructions  then AMPure cleaned and aliquoted and stored at  80 degrees celsius until further used. Binding Assay: RBP Halo fusions are in vitro transcribed and then translated with MegaScript T7 in vitro transcription kit ThermoFisher and Wheat Germ extract according to manufacturers instructions 50 ul in vitro translation reaction. The in vitro translated proteins are recovered using 25 ul Magne Halo Ligand beads Promega washed and resuspended in PBSN 1X PBS  0 005% NP 40 with added RNAse A and Turbo DNAse Thermo. post 1 hour of incubation  the beads are washed 4 times  twice with high salt PBSN + 1M MgCl2 and twice with normal salt PBSN. Beads are ruspended in 50 ul of PBSN and mixed with one aliquote of substrate RNA 150 ng in total brought to room temperature and diluted to a final volume of 50 ul of PBSN. The binding reaction occurs at room temperature for one hour rotating end over end subsequently 3 washes in PBSN are performed to remove unbound molecules and elution is perfomed in 15 ul of RNAse free water at 70 degrees celsius for 8 minutes resuspending the beads by pipeting up and down whilst the tube is in the thermocycler for a total of 3 times during the 8 minutes at 70 degrees. 11 ul of the eluted RNA are used to prepare an illumina compatible next generation sequencing library. Upon cell harvest  700 \u00b5L Qiazol QIAGEN was directly added onto the cells on ice and mixed. The cell extract was either stored at  80 \u00b0C or RNA extraction was continued immediately by adding 140 \u00b5L chloroform. This Qiazol/chloroform mixture was shaken for 30 sec and incubated at RT for 3 min  before centrifugation at 9.000 g for 5 min at 4 \u00b0C. The upper aqueous phase was transferred to a new tube and an equal volume of isopropanol was added. The tube was inverted 5 times followed by 10 min incubation at RT. The mixture was centrifuged at 9.000 g and 4 \u00b0C for 10 min and the supernatant discarded. The pellet was washed in 700 \u00b5L cold 70% ethanol and centrifuged at 15.000 g for 5 min at 4 \u00b0C. The supernatant was discarded entirely  and the pellet air dried for 5 minutes  before resuspension in nuclease free water. RNA concentration was determined by nanodrop Nanodrop 2000c. RNA was DNase treated using the Turbo DNase Kit Thermo Fisher for 30 min at 37 \u00b0C and the reaction was column purified using RNA Clean&Concentrator kit Zymo Research. The library preparation was carried out using the NEXTFLEX small RNA library preparation kit v3  PerkinElmer catalog # NOVA 5132 06 according to manufacturer instructions. The quality of every cDNA library was determined on an Agilent Bioanalyzer instrument according to the manufacturer's protocol.", "drHuRFISH rep2", "E MTAB 10834:drHuRFISH rep2", null, "isolate:not applicable|disease:normal|ENA FIRST PUBLIC:2023 08 11T00:23:05Z|organism:Danio rerio|organism:Danio rerio|ENA LAST UPDATE:2023 08 11T00:23:05Z|cell line:1|scientific name:Danio rerio|common name:zebrafish|organism part:liver|cell type:hepatocyte|genotype:wild type genotype|ENA FIRST PUBLIC:2023 08 11|ENA LAST UPDATE:2023 08 11", null, null, null, null, null, null, null, null, "NextSeq 550 paired end sequencing; RNA Affinity Purification followed by sequencing RAP seq allows identification of transcriptome wide RBP binding sites using recombinant RBPs and total purified cellular RNA", "E MTAB 10834:drHuRFISH rep2 p", "drHuRFISH rep2 p", "RNA Affinity Purification followed by sequencing RAP seq allows identification of transcriptome wide RBP binding sites using recombinant RBPs and total purified cellular RNA", "HepG2 were obtained from American Type Culture Collection ATCC  Rockville  MD. The cell line was mycoplasma free when periodically tested with Mycoplasmacheck Eurofins Genomics. HepG2 were cultured in T75 flasks at 37 \u00b0C and 5 % CO2 atmosphere using Dulbecco's Modified Eagle Medium DMEM supplemented with 1/100 Penicillin/Streptomycin P/S  Sigma and 10 % fetal bovine serum Hyclone  GE healthcare. HepG2 cells were maintained by splitting 1/6 three times a week  done by aspirating the medium  gently washing the cells with phosphate buffered saline PBS  without xxx+  Sigma and detaching them with 2 mL of a trypsin EDTA solution 0.05%  Sigma for 3 5 min. Trypsin EDTA was inactivated with a minimum of 10 fold surplus of culture medium before a cell fraction was passaged Substrate preparation: DNAse treated and column cleaned total RNA was fragmeted at 95 degrees celsius in Tris HCl 80 mM and MgCl2 8 mM final concentrations for 4 minutes and 30 seconds. post fragmentation  the RNA was cleaned using 1 volume of RNA CleanXP Ampure beads and 1 volume of isopropanol; post 2 80% Ethanol washes the RNA was eluted in half the volume of AMPure beads used. three prime dephosphorylation was then performed with T4PNK in TAM buffer Tris Acetate 50 mM  Magnesium Chloride 10 mM  pH8.5 at room temperature for 4 hours followed by AMPure clean up and subsequently five prime phosphorylated with T4PNK in T4PNK buffer with addition of ATP at 37 degreees celsius for 1 hour according to NEB manufacturer instructions  then AMPure cleaned and aliquoted and stored at  80 degrees celsius until further used. Binding Assay: RBP Halo fusions are in vitro transcribed and then translated with MegaScript T7 in vitro transcription kit ThermoFisher and Wheat Germ extract according to manufacturers instructions 50 ul in vitro translation reaction. The in vitro translated proteins are recovered using 25 ul  Magne Halo Ligand beads Promega washed and resuspended in PBSN 1X PBS  0 005% NP 40 with added RNAse A and Turbo DNAse Thermo. post 1 hour of incubation  the beads are washed 4 times  twice with high salt PBSN + 1M MgCl2 and twice with normal salt PBSN. Beads are ruspended in 50 ul of PBSN and mixed with one aliquote of substrate RNA 150 ng in total brought to room temperature and diluted to a final volume of 50 ul of PBSN. The binding reaction occurs at room temperature for one hour rotating end over end subsequently 3 washes in PBSN are performed to remove unbound molecules and elution is perfomed in 15 ul of RNAse free water at 70 degrees celsius for 8 minutes resuspending the beads by pipeting up and down whilst the tube is in the thermocycler for a total of 3 times during the 8 minutes at 70 degrees. 11 ul of the eluted RNA are used to prepare an illumina compatible next generation sequencing library.  Upon cell harvest  700 \u00b5L Qiazol QIAGEN was directly added onto the cells on ice and mixed. The cell extract was either stored at  80 \u00b0C or RNA extraction was continued immediately by adding 140 \u00b5L chloroform. This Qiazol/chloroform mixture was shaken for 30 sec and incubated at RT for 3 min  before centrifugation at 9.000 g for 5 min at 4 \u00b0C. The upper aqueous phase was transferred to a new tube and an equal volume of isopropanol was added. The tube was inverted 5 times followed by 10 min incubation at RT. The mixture was centrifuged at 9.000 g and 4 \u00b0C for 10 min and the supernatant discarded. The pellet was washed in 700 \u00b5L cold 70% ethanol and centrifuged at 15.000 g for 5 min at 4 \u00b0C. The supernatant was discarded entirely  and the pellet air dried for 5 minutes  before resuspension in nuclease free water. RNA concentration was determined by nanodrop Nanodrop 2000c. RNA was DNase treated using the Turbo DNase Kit Thermo Fisher for 30 min at 37 \u00b0C and the reaction was column purified using RNA Clean&Concentrator kit Zymo Research. The library preparation was carried out using the NEXTFLEX small RNA library preparation kit v3  PerkinElmer catalog # NOVA 5132 06 according to manufacturer instructions. The quality of every cDNA library was determined on an Agilent Bioanalyzer instrument according to the manufacturer's protocol.", "Experimental Factor: immunoprecipitate:anti drHuR|Experimental Factor: organism:Danio rerio", "RNA-Seq", "TRANSCRIPTOMIC", "other", "PAIRED", "ILLUMINA", "NextSeq 550", null, "ERP131171", "NextSeq 550 paired end sequencing; RNA Affinity Purification followed by sequencing RAP seq allows identification of transcriptome wide RBP binding sites using recombinant RBPs and total purified cellular RNA", "ENA FIRST PUBLIC:2023 08 11|ENA LAST UPDATE:2023 08 11", "drHuRFISH_rep2.R1.fastq.gz drHuRFISH_rep2.R2.fastq.gz", "fastq fastq", 2408024386.0, 29012342.0, "E MTAB 10834:drHuRFISH rep2.R", "0:40 1:43", "A:566287649;C:627743498;G:663052609;T:550604982;N:335648", 40, 43, null, null, 566287649, 627743498, 663052609, 550604982, 335648, "ERX6101492", "ERS7377022", "ERA5607540", "Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, Sweden|European Nucleotide Archive", "Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, Sweden|European Nucleotide Archive", 2, 0.68524, 0.62721, 0.16916, 0.1656, 0.93501, 0.94541, 0.7028, 0.64738, 40, 43, "B", "B", "biological fallback assumption", "illumina", "nextseq", "unknown", "small_rna", "unknown", "bulk", "unknown", "unknown", null, "Sweden", "2023-08-11", "Undetermined", "Undetermined", "Multi-tissue", "Multi-system"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["10210"], "units": {}, "query_ms": 12.644997004827019}