HeLa culture, transfection, and labeling of Halo-fusion proteins
By savannah onThe authors developed a protocol to investigate the role of the NEMO in Parkin-dependent mitochondrial clearance. Halo-fusion constructs, including NEMO and OPTN used in the study, allowed the authors to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to the characterization of NEMO’s involvement in mitophagy.
Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling
By savannah onThe authors approached image analysis in a multitude of creative, effective ways, and importantly the authors maintained consistency of analysis within experiments in order to present the results with integrity and reproducibility.
Immunostaining of iPSC-derived neurons for quantification of synaptic proteins
By savannah onHere, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy
By savannah onThree standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to 1,6-Hexanediol. This protocol describes the authors’ approach to investigating standards B and C.
Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
By savannah onThe live imaging of NEMO occupancy on damaged mitochondria was a necessary complement to the authors’ parallel fixation studies. Their reporting of these results would not have been possible without real-time, live cell imaging.
Live-cell imaging for synaptic vesicle precursors in human iNeuron axons
By savannah onThe authors describe procedure and equipment used for live-imaging of synaptic vesicle precursors. This was performed using DIV21 human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on scheduled upgrades to microscopy equipment during the course of this study.
Microscopy-based bead protein-protein interaction assay
By savannah onThis protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.
Gene editing of YIPF4 in hESCs V3
By savannah onThis protocol describes the creation of YIPF4 knockout cell lines in H9 hESC cells using CRISPR-Cas9. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342
Characterizing spatial and temporal properties of ER-phagy receptors
By savannah onAssociated with preprint: https://doi.org/10.1101/2023.06.26.546565
Analysis of ER structures in Cultured Induced Neuron axons
By savannah onAssociated with preprint: https://doi.org/10.1101/2023.06.26.546565
Analysis of ER Flux in Cultured Induced Neurons using Keima ER reporters
By savannah onAssociated with preprint: https://doi.org/10.1101/2023.06.26.546565
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.
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.
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.
Isolation of NeuN+ cells from brain tissue (for CUT and RUN)
By savannah onThis protocol describes the steps to isolate NeuN+ cells from brain tissue in preparation for CUT and RUN.
Single cell/nuclei RNAseq analysis
By savannah onThis protocol describes the process for the single cell/nuclei RNA sequencing data of the manuscript, "L1retrotransposons drive human neuronal transcriptome complexity and functional diversification," from fetal forebrain and adult prefrontal cortex tissue.
Image processing of full-length monomeric LRRK2
By savannah onThis protocol assumes that 2000 to 4000 movies of full-length LRRK2 embedded on vitreous ice are collected with an electron microscope equipped with a direct detector. It focuses primarily on the monomeric population of LRRK2 and has been leading to 3-4 Å resolution structures.
LRRK1 expression and purification
By savannah onProtein purification protocol for full-length LRRK1 as done by Leschziner and Reck-Peterson Labs. Original protocol by Janice M. Reimer and Yu Xuan Lin.
Quick guide to use paceTOMO for cryo-ET data collection from Titan Krios
By savannah onThis quick guide provides key minimal steps for preparing the Titan/SerialEM for the tomogram data collection on lamella or in vitro specimens with a K3 camera. paceTOMO routine is also included for a typical tomogram data collection session. Please note that this is not an exhaustive guide, but summarizes the order of key steps.