{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH11.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH05.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH04.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH02.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH02.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH06.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH04.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH06.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH11.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH05.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH08.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH03.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_STIM_pH08.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/20221216_E1_RM_JAL_0489_NON_pH03.raw"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD070784/txt.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["j.vanloosdregt@umcutrecht.nl"],"submitter":["Harmjan Vos"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Monocytes  activation-induced alternative splicing isoforms"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD070784"],"tissue":["Monocyte","Blood"],"sample_protocol":["Proteins were denatured and alkylated in 50 µl 8 M Urea, 1 M ammonium bicarbonate (ABC) containing 10 mM TCEP (tris (2-carboxyethyl) phosphine hydrochloride) and 40 mM 2-chloro-acetamide and separated from the beads. After 4-fold dilution with 1 M ABC and digestion with trypsin (20 µg)(Promega), peptides were desalted with homemade C-18 stage tips (3 M, St Paul, MN), eluted with 80% Acetonitrile (ACN) and, after evaporation of the solvent in the speedvac, redissolved in Britton & Robinson buffer composed of 20 mM acetic acid, 20 mM phosphoric acid, and 20 mM boric acid titrated with NaOH to pH 11 for Sax fractionation and loaded on a stage tip with SAX material (3 M, St Paul, MN). 7 Fractions (Ph= 11,8,6,5,4,3,2) of 50 microliter were loaded subsequently on a c18 stage tip after acidification with 20 microliter 20% Formic acid. Peptides were eluted with buffer B (80% ACN, 0.1% FA) and dried in the speedvac  and resolved in A (0,1% formic acid). After separation on a 30 cm pico-tip column (75 µm ID, New Objective) in-house packed with C-18 material (1.9 µm aquapur gold, dr. Maisch) using a 270 minute gradient (7% to 80% ACN, 0.1% FA), delivered by an easy-nLC 1200 (Thermo), peptides were  electro-sprayed directly into a Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Scientific), that was run in DDA mode with a cycle time of 1 second, with the FAIMS compensation voltage (CV) switching between -45V and -65V. The full scan (400-1400 mass range) was performed at a resolution of 240,000. Ions reaching an intensity threshold of 10E4, were isolated by the quadrupole with an 0.7 Da window, fragmented with a HCD collision energy of 30% and measured in de Iontrap in rapid mode with a maximuminjection time of 40 milliseconds."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["After splitting of the raw files into the two CV channels with Freestyle Software (Thermo), analysis of the raw data was performed with MaxQuant (Version 2.6.4.0), using the standard settings and the Uniprot database for Homo sapiens (Dec 2021) and a monocyte specific with the predicted protein sequences from our novel transcriptome. Oxidation of Methionine, and Acetylation of protein N-termini were set as variable modifications while Carbamidomethylation (C) was set as a fixed modification."],"omics_type":["Proteomics"],"labhead":["Jorg van Loosdregt"],"instrument_platform":[""],"labhead_affiliation":["Center for Translational Immunology, University Medical Center Utrecht and Utrecht University, Utrecht, the Netherlands"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["h.r.vos-3@umcutrecht.nl"],"publication":["Not available"],"submitter_affiliation":["University Medical Center Utrecht\nCenter for Molecular Medicine"],"submitter_country":["Netherlands"],"additional_accession":[]},"is_claimable":false,"name":"Native long-read RNA sequencing of human monocytes reveals activation-induced alternative splicing toward functional isoforms","description":"Alternative splicing is a key mechanism for expanding transcriptomic and proteomic complexity, yet its role in innate immune activation remains incompletely understood. Here, we applied Oxford Nanopore native RNA-sequencing to generate an isoform-level transcriptome of primary human monocytes before and after activation with lipopolysaccharide. We identify over 24,000 expressed isoforms, including thousands of previously unannotated variants. Activation induced widespread isoform-specific expression changes, leading to extensive isoform switching events, validated using matched short-read RNA-Seq. These activation-induced shifts enhanced transcript immune-regulatory functions: activated monocytes preferentially express longer, coding-competent isoforms with complete open reading frames, fewer retained introns, and increased domain complexity. By integrating matched Ribo-seq and proteomic data, we demonstrate that these isoform modulations are associated with enhanced translation of immune effector proteins. Together, our findings position alternative splicing as a dynamic and functional regulator of monocyte activation, emphasizing the need for isoform-level resolution to fully understand immune cell function and inflammation.","dates":{"publication":"2026-04-29","submission":"2025-11-16"},"accession":"PXD070784","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:1589","NEWT:135622","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:383379","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:244366","NEWT:1246791","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2853422","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:109760","NEWT:29031","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:470","NEWT:5911","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:6493","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:8655","NEWT:5147","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT:443906","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:214053","NEWT:80863","NEWT:90675","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:8695","NEWT:884204","NEWT:1123869","NEWT:9544","NEWT:9545","NEWT:979","NEWT:7370","NEWT:83906","NEWT:1134506","NEWT:255470","NEWT:38783","NEWT:6426","NEWT:33090","NEWT:9935","NEWT:287889","NEWT:305959","NEWT:13489","NEWT:92867","NEWT:92866","NCBITaxon:3055","NEWT:51750","NEWT:202950","NEWT:295027","NCBITaxon:11320","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:90690","NEWT:1436183","NEWT:4232","NEWT:416348","NEWT:11298","NEWT:4236","NEWT:7742","NEWT:196627","NEWT:200308","NEWT:200302","NEWT:870435","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:2666255","NEWT:999810","NCBITaxon:5693","NEWT:28995","NEWT:1392998","NEWT:380394","NEWT:226900","NEWT:1266738","NEWT:231490","NEWT:244704","NEWT:7725","NEWT:9909","NEWT:9902","NEWT:430615","NEWT:72664","NEWT:563041","NEWT:36599","NEWT:41949","NEWT:326423","NEWT:452467","NEWT:198822","NEWT:36111","NEWT:326424","NEWT:1678078","NEWT:749906","NEWT:418985","NEWT:749907","NEWT:15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