[IDF] Investigation Title RNA-seq analysis of Brpf1 knockout versus wild-type neurons derived from BALB/c mouse brain [49] Experiment Description "This study investigates the transcriptional impact of targeted knockout (KO) of the Brpf1 gene in primary mouse neurons using RNA sequencing (RNA-seq). Brain tissue was dissected from four BALB/c mice. Neuronal cells were isolated and cultured in vitro for two weeks prior to RNA extraction. A targeted knockout of Brpf1 was introduced in two samples, while two samples were maintained as wild-type controls, resulting in two biological replicates per condition. Total RNA was extracted and mRNA libraries were prepared by the EMBL Genomics Core Facility. Sequencing was performed on the Illumina HiSeq 4000 platform using paired-end mode. This dataset enables comparative transcriptomic analysis between Brpf1 knockout and wild-type neurons to assess the gene’s role in neuronal gene regulation." Comment[AEExperiment] Comment[LabID_uuid] 114c10ae-9302-4a3a-b6c9-86af435dac7f Experimental Design biological replicate genetic modification design Experimental Design Term Source REF EFO EFO Experimental Design Term Accession Number EFO_0002091 EFO_0001758 Experimental Factor Name genotype genetic modification a genotype genetic modification Experimental Factor Term Source REF EFO EFO Experimental Factor Term Accession Number EFO_0000513 EFO_0000510 Person Last Name trainee49 Person First Name trainee49 Person Middle Name Person Email trainee49@fake.org Person Affiliation European Molecular Biology Laboratory Person Roles submitter;submitter;co-investigator Public Release Date 08/04/2027 Protocol Name Standard HiSeq 4000 paired-end Sequencing Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina Protocole Type nucleic acid sequencing protocol growth protocol growth protocol nucleic acid extraction protocol nucleic acid library construction protocol Protocol Description paired-end sequencing on illumina HiSeq 4000 "Mouse brains were dissected and processed to obtain primary neuronal cells. Tissue was enzymatically digested and mechanically dissociated into a single-cell suspension. Cells were filtered, counted, and prepared for downstream culture." Primary neurons were cultured for two weeks under controlled conditions. Cells were maintained in specialized medium with regular partial medium changes. Culture conditions supported neuronal maturation and network formation. A targeted knockout of Brpf1 was introduced using CRISPR-Cas9 (KO samples). Neuronal cultures were transfected with sgRNA constructs targeting the gene. Edited cells were maintained for downstream transcriptomic analysis. "RNA was extracted from ~10 million cells using the RNeasy Mini kit (Qiagen) and DNase digestion was performed using TURBO DNase (Thermo Fisher Scientific), according to the manufacturer’s instructions and an additional clean-up of the RNA was then performed using the RNeasy Mini kit (Qiagen). Quality of the RNA was assessed on Bioanalyzer using the Agilent RNA 6000 Nano Kit and samples with a RNA integrity number (RIN) of 10 were used for library generation." "Library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, in combination with the NEBNext Poly(A) mRNA Magnetic Isolation Module for mRNA enrichment, according to the manufacturer’s instructions." Protocol Hardware HiSeq 4000 Protocol Term Source REF EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0004170 EFO_0005518 EFO_0005518 EFO_0002944 EFO_0004184 Protocol Comment[LabID_uuid] 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 a3462203-b867-4e5e-85d2-2c69ba445d53 3121a3cb-a95f-4286-aa72-3b178888f54e 5ade9bae-ef6d-43da-a7f8-c3eb35bc88bd f43973f8-0f6a-4739-846b-215adba2d662 Term Source Name MGED Ontology ArrayExpress EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php https://www.ebi.ac.uk/biostudies/arrayexpress http://www.ebi.ac.uk/efo/ [SDRF] Source Name Comment[Sample_LabID_uuid] Material Type Term Source REF Characteristics[age] Characteristics[cell type] Characteristics[developmental stage] Characteristics[genetic modification] Characteristics[genotype] Characteristics[initial time point] Characteristics[organism part] Characteristics[sex] Characteristics[strain] Protocol REF Protocol REF Protocol REF Protocol REF Extract Name Material Type Term Source REF 2 Comment[Dataset_LabID_uuid] Comment[BARCODE] Comment[LIBRARY_LAYOUT] Comment[LIBRARY_SOURCE] Comment[LIBRARY_STRATEGY] Comment[LIBRARY_SELECTION] Comment[QUALITY_SCORING_SYSTEM] Comment[LIBRARY_ENCODING] Comment[ASCII_OFFSET] Comment[ORIENTATION] Protocol REF Performer Assay Name Comment[Assay_LabID_uuid] Technology Type Array Data File Comment[MD5] Factor Value[genetic modification] Factor Value[genotype] 49_KO_Rep1 e985b44d-341c-465f-8622-0b0930c830ce # TODO MGED Ontology 21 weeks Neuron adult gene knock out Brpf1- birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_KO_Rep1 cell MGED Ontology b01d7421-d574-4f19-9337-0e65836c777c CCC PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_3_1_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 gene knock out Brpf1- 49_KO_Rep1 e985b44d-341c-465f-8622-0b0930c830ce # TODO MGED Ontology 21 weeks Neuron adult gene knock out Brpf1- birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_KO_Rep1 cell MGED Ontology b01d7421-d574-4f19-9337-0e65836c777c CCC PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_3_2_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 gene knock out Brpf1- 49_KO_Rep2 59ec8ab5-8fe9-4a72-805d-779b3ecd289e # TODO MGED Ontology 20 weeks Neuron adult gene knock out Brpf1- birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_KO_Rep2 cell MGED Ontology d87b1df1-7d4c-4b39-9872-a37126a53805 GGGG PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_4_1_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 gene knock out Brpf1- 49_KO_Rep2 59ec8ab5-8fe9-4a72-805d-779b3ecd289e # TODO MGED Ontology 20 weeks Neuron adult gene knock out Brpf1- birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_KO_Rep2 cell MGED Ontology d87b1df1-7d4c-4b39-9872-a37126a53805 GGGG PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_4_2_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 gene knock out Brpf1- 49_WT_Rep1 6265df73-e324-4bc7-9845-4c0171b6bafc # TODO MGED Ontology 23 weeks Neuron adult none wild type birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_WT_Rep1 cell MGED Ontology d66e105a-8c5b-425d-a516-6849537a830b AA PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_1_1_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 none wild type 49_WT_Rep1 6265df73-e324-4bc7-9845-4c0171b6bafc # TODO MGED Ontology 23 weeks Neuron adult none wild type birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_WT_Rep1 cell MGED Ontology d66e105a-8c5b-425d-a516-6849537a830b AA PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_1_2_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 none wild type 49_WT_Rep2 ed1e094e-45a8-4aa6-a401-d8d7ba0546cc # TODO MGED Ontology 22 weeks Neuron adult none wild type birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_WT_Rep2 cell MGED Ontology 10d7db2c-3cfc-4fe1-875a-4ae37c2578dc TT PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_2_1_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 none wild type 49_WT_Rep2 ed1e094e-45a8-4aa6-a401-d8d7ba0546cc # TODO MGED Ontology 22 weeks Neuron adult none wild type birth brain unknown sex BALB/c Neuron Isolation from Mouse Brain Primary Neuron Culture with Targeted Knockout of Brpf1 RNA Extraction with RNeasy Mini kit mRNA Library Preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina 49_WT_Rep2 cell MGED Ontology 10d7db2c-3cfc-4fe1-875a-4ae37c2578dc TT PAIRED TRANSCRIPTOMIC RNA-Seq Oligo-dT phred ascii @ 5'-3'-3'-5' Standard HiSeq 4000 paired-end Sequencing USER trainee49 XYZ_PE_Man_4_1_trainee49 2588de18-b61b-4b4d-a9f4-2fabd5488fe3 sequencing assay XYZ_PE_Man_4_1_trainee49_Sequence_file_XYZ_PE_Man_4_1_trainee49_2_2_sequence.txt.gz b9571b5fafe80a187b35cdc85c9b3082 none wild type