Long-read RNA seq analysis using Talon
By savannah onThis is a snakemake pipeline that takes Oxford Nanopore Sequencing (ONT) data (fastq) as input, generates fastq stats using nanostat, performs fastq processing and filtering using pychopper, maps the reads to the genome using minimap2, and uses talon to assemble and quantify transcripts. It is forked from ANNSeq. The link includes the dag of the pipeline.
pCAG-TSF-ATG13 (363-517)
By savannah onMammalian expression of TwinStrep-Flag tagged ATG13 (363-517).
Plasmid: V5-KANSL1 pLVX-EF1α-IRES-Puro
By savannah onHuman KANSL1 cDNA with N-terminal V5 tag cloned into the pLVX-EF1α-IRES-Puro plasmid (EcoRI and SpeI restriction sites) from Clontech, Takara Bio.
pCAG-TSF-ATG13 (450-517)-ULK1 (836-1050)
By savannah onMammalian expression of ATG13 C-terminal and ULK1 MIT domain fusion complex.
pGEX-4T1-GST-Thrombin-TEV-EGFP-hNEMO
By savannah onHuman p62 with silent mutation for sip62 #5 (Thermo Scientific). Internal reference: SMC516 Plasmid for the expression of GFP labelled NEMO (SMC Internal No. 1977).
Primary neuron culture for live imaging of axonal cargoes
By savannah onThis protocol describes the preparation and culture of mouse primary cortical neurons for live-imaging experiments. Neurons were transfected 16-24 hours before imaging using Lipofectamine 2000.
RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors
By savannah onFor quantitative results, gray cells represent breaks between independent experiments, or between individual fields of view. Please contact E.L.F. Holzbaur (corresponding author, [email protected]) with any questions or concerns.
RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors
By savannah onPublished: Gain-of-function mutations in the LRRK2 gene cause Parkinson’s disease (PD), characterized by debilitating motor and non-motor symptoms. The authors’ results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD. View original preprint.
RNA extraction and quantitative PCR to assay inflammatory gene expression
By savannah onThe authors developed this protocol to test levels of NF-kB response genes in a cell model transiently over-expressing Parkin. With this technique the authors found significant upregulation of key pro-inflammatory genes normalized to a housekeeping gene, Gapdh.
Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore
By savannah onIn a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12--ATG5-ATG16L1 machinery establishes a high level of regulatory control.
Plasmid: pLV[Exp]-EF1A>V5/hKAT8[NM_032188.3]:IRES:Puro
By savannah onHuman KAT8 gene with N-terminal V5 tag cloned into the pLV plasmid backbone under a EF1a promoter and including IRES puromycin selection cassette by Vectorbuilder.
Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore: Molecular dynamics simulation data
By savannah onAtomistic molecular dynamics simulation data set accompanying manuscript, "Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore.”
Leucine-rich repeat kinase 2 at a glance
By savannah onThis Cell Science at a Glance article (and the accompanying poster) deliver a snapshot of the authors’ current understanding of LRRK2 function, dysfunction, and links to disease. Journal of Cell Science (2023) 136, jcs259724 (pending publication).
Bulk RNA sequencing analysis
By savannah onThis protocol describes the steps for the bioinformatical analysis of bulk RNA sequencing with a focus on evolutionary young L1s.
Iso-Seq mapping to L1HS/PA2 consensus sequence
By savannah onThe protocol describes the steps to map HiFi reads to a consensus sequence and retrieve density plots.
Lentivirus plasmids for sgRNA: pLV[Exp]-U6>NT-Seq1-hPGK>mApple
By savannah onPlasmid vector encoding a non-targeting sgRNA sequence under a U6 promoter and a mApple fluorescent reporter. Generated by Vectorbuilder in the pLV backbone
