{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":[null],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14537"],"description":["SLAM-sequencing was performed on i3 LMNs following protocols adapted from Herzog et al. (2017). Three biological replicates of i3 LMNs were treated with 100 µM 4sU (Control) + labelling medium or 100 µM 4sU + BDNF (BDNF condition) labelling medium on Day 7 for 1h, 2h, and 6h. Cells were washed with PBS and RNA was extracted using the Qiagen RNeasy isolation and purification kit. RNA concentration was measured, and after equilibrating the amount of RNA in each sample, iodoacetamide (10 mM), NaPOH4 (pH 8, 50 mM), and DMSO (50% v/v) were added to each sample and incubated at 50 C for 15 min to facilitate the thiol-alkylation of 4sU. The samples were processed on RNeasy columns to re-isolate RNA. Sequencing libraries were prepared using the KAPA HyperPrep Kit with RiboErase kit (Roche). Samples were sequenced at 2 x 100 base pairs on an Illumina NextSeq 2000 machine."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - The human induced pluripotent stem cells used are the WTC11 line, stably expressing a doxycycline-inducible hNIL construct and CRISPR/cas9. Briefly, iPSCs were seeded on tissue culture dishes coated with 120-180 μg/ml Geltrex (Thermo Fisher). Cells were maintained in iPSC medium containing Essential 8 Flex Medium (E8F, Thermo Fisher), E8 Flex supplement (Thermo Fisher), and 100 U/ml Pen/Strep and maintained in a 37 ºC, 5% CO2 incubator. Medium change was performed every 1-2 days. Passage of cells were performed with Stem Pro Accutase (Thermo Fisher) for 1-2 min at 37 ºC. Accutase was removed by centrifugation at 300 g for 5 min, and the cells were reseeded in iPS cell media supplemented with 10 μM ROCK inhibitor (Y-27632, Selleckchem) to facilitate survival. ROCK inhibitor was removed after 24 h.","Nucleic Acid Extraction - Cells were washed with PBS and RNA was extracted using the Qiagen RNeasy isolation and purification kit. RNA concentration was measured, and after equilibrating the amount of RNA in each sample, iodoacetamide (10 mM), NaPOH4 (pH 8, 50 mM), and DMSO (50% v/v) were added to each sample and incubated at 50 C for 15 min to facilitate the thiol-alkylation of 4sU.","Sequencing - Samples were sequenced at 2 x 100 base pairs on an Illumina NextSeq 2000 machine.","Library Construction - Sequencing libraries were prepared using the KAPA HyperPrep Kit with RiboErase kit (Roche)."],"figure_sub":["MINSEQE Score","Assays and Data","Processed Data","organisation","MAGE-TAB Files"],"data_protocol":["Data Transformation - Samples were aligned to the Genome Reference Consortium Human Build 38 (GRCh38) genome using hierarchical indexing for spliced alignment of transcripts - 3 nucleotides (HISAT-3N) with gene models from GENCODE v34 (Frankish et al., 2019; Zhang et al., 2021)(Y. Liao et al., 2014) and quantified using FeatureCounts using gene models from GENCODE v34(Y. Liao et al., 2014) For the differential expression analysis of the total RNA, all samples were run using the standard DESeq2(Love et al., 2014) workflow in R."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["RiboErase Roche","NA","Qiagen RNaesy kit","NextSeq 2000"],"study_type":["RNA-seq of total RNA"],"species":["Homo sapiens"],"additional_accession":["ERP165141"],"pubmed_authors":["Anna-Leigh Brown"]},"is_claimable":false,"name":"RNA-sequencing using SLAM-sequencing protocol of human iPSC-derived lower motor neurons treated with BDNF for 1, 2, 6 h","description":"SLAM-sequencing was performed on i3 LMNs following protocols adapted from Herzog et al. (2017). Three biological replicates of i3 LMNs were treated with 100 µM 4sU (Control) + labelling medium or 100 µM 4sU + BDNF (BDNF condition) labelling medium on Day 7 for 1h, 2h, and 6h. Cells were washed with PBS and RNA was extracted using the Qiagen RNeasy isolation and purification kit. RNA concentration was measured, and after equilibrating the amount of RNA in each sample, iodoacetamide (10 mM), NaPOH4 (pH 8, 50 mM), and DMSO (50% v/v) were added to each sample and incubated at 50 C for 15 min to facilitate the thiol-alkylation of 4sU. The samples were processed on RNeasy columns to re-isolate RNA. Sequencing libraries were prepared using the KAPA HyperPrep Kit with RiboErase kit (Roche). Samples were sequenced at 2 x 100 base pairs on an Illumina NextSeq 2000 machine.","dates":{"release":"2025-05-30T00:00:00Z","modification":"2026-06-03T12:46:21.983Z","creation":"2024-10-15T13:26:34.497Z"},"accession":"E-MTAB-14537","cross_references":{"ENA":["ERP165141"],"EFO":["EFO_0002944","EFO_0004170","EFO_0009653","EFO_0005518","EFO_0003816","EFO_0004184"]}}