DOI: 10.6084/m9.figshare.13203725.v3
title: Cells treated with sgRNA against CALM

description: <b>Introduction</b> We created this dataset to provide sample movies for the paper, “<b><i>Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics</i></b>”, https://doi.org/10.1073/pnas.2020346117. In this paper, CRISPR-mediated protein knock-down was used to study the functional role of 67 proteins during clathrin-mediated endocytosis. Due to the extremely large data volume, we only provide a small subset of movies for 2 conditions. The full dataset (or part of it) is available upon request. Please contact Marcel Mettlen (marcel.mettlen@utsouthwestern.edu). The movies provided are fluorescent images of ARPE19 cells expressing eGFP-CLC treated with sgRNA against CALM. The corresponding scramble controls are also included. Readers are referred to the software pipeline cmeAnalysis to analyze these datasets. The pipeline and instructions are available on GitHub: https://github.com/DanuserLab/cmeAnalysis <b>Methods and Materials</b> For a detailed description of cell generation, sgRNA production and cell transduction, readers are referred to the main publication and the supporting material (10.1073/pnas.2020346117​.) TIR-FM imaging On the day of TIR-FM imaging, cells were checked for appropriate spreading and density. Fresh media was added at least 30 min before imaging each slide-mounted coverslip. To minimize experiment-to-experiment variation during image acquisition, all microscope components were primed by a 30-min mock acquisition: pre-determined laser and camera settings were used for acquisition without a sample on stage. These settings were defined prior to the screen to allow for sufficient signal intensity over background while keeping photobleaching &lt;10% over the course of the acquisition time. It is important to image with the highest possible intensity, while still avoiding photobleaching. During imaging, cells were maintained at 37°C in the same culture media. Two TIR-FM systems were used for acquisition of live cell data: #1 fully motorized Nikon Eclipse Ti-E inverted microscope with integrated second-generation Perfect Focus System, 60x Nikon 1.49NA TIRF DIC objective, coupled to an Andor “Diskovery TIRF/ Borealis widefield illuminator” equipped with an additional 1.8x tubelens (yielding a final magnification of 108x), motorized laser incident angle adjustment, set to 80 nm penetration depth, and an Andor laser launch. Images were acquired with a PCO-Edge 16 bit, 2560x2160px sCMOS camera. Temperature was maintained via an OKO lab custom built full body environmental chamber with temperature control and CO2 stage incubator operated by Bold Line controller and OKO-Touch with SmartBox for data logging. Components were controlled via MetaMorph v.7.7 (Molecular Devices). TIR-FM system #2 is closely related, but uses an Andor Zyla 4.2 16 bit, 2048x2048 px sCMOS camera. Time-lapse movies for all conditions were acquired for 7.5 min with a 1 sec interval between consecutive frames. Given the storage size of a single movie (up to 3.6Gb), image stacks were split into two along the largest xy dimension using a custom-written Fiji macro, yielding a total of 22-24 datasets per condition.