Visualization of loop extrusion by DNA nanoscale tracing in single cells

SARS-COV-2 drug repurposing - Caco2 cell line

Large-scale electron microscopy database for human type 1 diabetes

RNA localisation

CRISPR

Brain

Image analysis code

Sequencing data

Project website

Experiment A

Screen B

Stitched max-projected fluorescent confocal images

Adult mouse corpus callosum

Drosophila endoderm

AC16s human cardiomyoctye cells

Bronchial epithelial cell culture

stable overexpression of HIST1H2BJ-mCherry and LMNA

Jurkat E6.1 transfected with emerald-VAMP7

Homozygous GFP integration into mitotic genes

Plate-bound anti-CD3 activation

2-(9-oxoacridin-10-yl)acetic acid

cridanimod

Time

Genotype

Light exposure

Homo sapiens

Arabidopsis thaliana

Danio rerio

human

thale cress

zebrafish

http://purl.obolibrary.org/obo/NCBITaxon_9606

http://purl.obolibrary.org/obo/NCBITaxon_3702

http://purl.obolibrary.org/obo/NCBITaxon_7955

Cells were cultured on poly-L-lysine treated coverslips. Culture media was aspirated, and coverslips were washed once with PBS. Cells were fixed by incubating for 10 min with 4 % formaldehyde/PBS, washed twice with PBS, and permeabilized by incubating (>3 h, -20°C) in 70 % ethanol. Cells were rehydrated by incubating (5 min, RT) with FISH wash buffer (10 % formamide, 2x SSC). For hybridization, coverslips were placed cell-coated side down on a 48μl drop containing 100 nM Quasar570-labelled probes complementary to one of REV-ERBα, CRY2, or TP53 transcripts (Biosearch Technologies) (see Table S6 for probe sequences), 0.1 g/ml dextran sulfate, 1 mg/ml E. coli tRNA, 2 mM VRC, 20 μg/ml BSA, 2x SSC, 10 % formamide and incubated (37°C, 20 h) in a sealed parafilm chamber. Coverslips were twice incubated (37°C, 30 min) in pre-warmed FISH wash buffer, then in PBS containing 0.5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) (5 min, RT), washed twice with PBS, dipped in water, air-dried, placed cell-coated side down on a drop of ProLong Diamond Antifade Mountant (Life Technologies), allowed to polymerize for 24 h in the dark and then sealed with nail varnish.

Immunostained spreads of Arabidopsis pachytene cells were prepared for 3D-SIM imaging as follows. To roughly stage the meiocytes, a single anther from a floral bud was removed and squashed in a drop of water on a clean slide under a coverslip and inspected using brightfield microscopy. Early- and mid-pachytene meiocytes were still stuck together within a meiocyte column, whilst late-pachytene meiocytes had begun to break apart from one-another. More precise staging of early and late pachytene meiocytes was also based on previously defined HEI10 behaviour, with mid pachytene meiocytes exhibiting an intermediate phenotype. The remaining 5 anthers containing meiocytes of the desired stage were dissected from the staged buds. They were then macerated using a brass rod on a No. 1.5H coverslip (Marienfeld) in 10 µl digestion medium (0.4% cytohelicase, 1.5% sucrose, 1% polyvinylpyrolidone in sterile water) for 1 min. Coverslips were then incubated in a moist chamber at 37 °C for 4 min before adding 10 µl of 2% lipsol solution followed by 20 µl 4% paraformaldehyde (pH 8). Coverslips were dried in the fume hood for 3 h, blocked in 0.3% bovine serum albumin in 1x phosphate-buffered saline (PBS) solution and then incubated with primary antibody at 4 °C overnight and secondary antibody at 37 °C for 2 h. In between antibody incubations, coverslips were washed 3 times for 5 min in 1x PBS plus 0.1% Triton X-100. Coverslips were then incubated in 10 µl DAPI (10 µg/ml) for 5 min, washed and mounted on a slide in 7 µl Vectashield.

Cells grown on coverslips were fixed in ice-cold methanol at _20 _ C for 10 min. After blocking in 0.2% gelatine from cold-water fish (Sigma) in PBS (PBS/FSG) for 15 min, coverslips were incubated with primary antibodies in blocking solution for 1h. Following washes with 0.2% PBS/FSG, the cells were incubated with a 1:500 dilution of secondary antibodies for 1 h (donkey anti- mouse/rabbit/goat/sheep conjugated to Alexa 488 or Alexa 594; Molecular Probes or donkey anti-mouse conjugated to DyLight 405, Jackson ImmunoResearch). The cells were counterstained with 1 _g ml_1 Hoechst 33342 (Sigma) to visualize chromatin. After washing with 0.2% PBS/FSG, the coverslips were mounted on glass slides by inverting them into mounting solution (ProLong Gold antifade, Molecular Probes). The samples were allowed to cure for 24-48 h.

Cells were cultured on poly-L-lysine treated coverslips. Culture media was aspirated, and coverslips were washed once with PBS. Cells were fixed by incubating for 10 min with 4 % formaldehyde/PBS, washed twice with PBS, and permeabilized by incubating (>3 h, -20°C) in 70 % ethanol. Cells were rehydrated by incubating (5 min, RT) with FISH wash buffer (10 % formamide, 2x SSC). For hybridization, coverslips were placed cell-coated side down on a 48μl drop containing 100 nM Quasar570-labelled probes complementary to one of REV-ERBα, CRY2, or TP53 transcripts (Biosearch Technologies) (see Table S6 for probe sequences), 0.1 g/ml dextran sulfate, 1 mg/ml E. coli tRNA, 2 mM VRC, 20 μg/ml BSA, 2x SSC, 10 % formamide and incubated (37°C, 20 h) in a sealed parafilm chamber. Coverslips were twice incubated (37°C, 30 min) in pre-warmed FISH wash buffer, then in PBS containing 0.5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) (5 min, RT), washed twice with PBS, dipped in water, air-dried, placed cell-coated side down on a drop of ProLong Diamond Antifade Mountant (Life Technologies), allowed to polymerize for 24 h in the dark and then sealed with nail varnish.

Immunostained spreads of Arabidopsis pachytene cells were prepared for 3D-SIM imaging as follows. To roughly stage the meiocytes, a single anther from a floral bud was removed and squashed in a drop of water on a clean slide under a coverslip and inspected using brightfield microscopy. Early- and mid-pachytene meiocytes were still stuck together within a meiocyte column, whilst late-pachytene meiocytes had begun to break apart from one-another. More precise staging of early and late pachytene meiocytes was also based on previously defined HEI10 behaviour, with mid pachytene meiocytes exhibiting an intermediate phenotype. The remaining 5 anthers containing meiocytes of the desired stage were dissected from the staged buds. They were then macerated using a brass rod on a No. 1.5H coverslip (Marienfeld) in 10 µl digestion medium (0.4% cytohelicase, 1.5% sucrose, 1% polyvinylpyrolidone in sterile water) for 1 min. Coverslips were then incubated in a moist chamber at 37 °C for 4 min before adding 10 µl of 2% lipsol solution followed by 20 µl 4% paraformaldehyde (pH 8). Coverslips were dried in the fume hood for 3 h, blocked in 0.3% bovine serum albumin in 1x phosphate-buffered saline (PBS) solution and then incubated with primary antibody at 4 °C overnight and secondary antibody at 37 °C for 2 h. In between antibody incubations, coverslips were washed 3 times for 5 min in 1x PBS plus 0.1% Triton X-100. Coverslips were then incubated in 10 µl DAPI (10 µg/ml) for 5 min, washed and mounted on a slide in 7 µl Vectashield.

Cells grown on coverslips were fixed in ice-cold methanol at _20 _ C for 10 min. After blocking in 0.2% gelatine from cold-water fish (Sigma) in PBS (PBS/FSG) for 15 min, coverslips were incubated with primary antibodies in blocking solution for 1h. Following washes with 0.2% PBS/FSG, the cells were incubated with a 1:500 dilution of secondary antibodies for 1 h (donkey anti- mouse/rabbit/goat/sheep conjugated to Alexa 488 or Alexa 594; Molecular Probes or donkey anti-mouse conjugated to DyLight 405, Jackson ImmunoResearch). The cells were counterstained with 1 _g ml_1 Hoechst 33342 (Sigma) to visualize chromatin. After washing with 0.2% PBS/FSG, the coverslips were mounted on glass slides by inverting them into mounting solution (ProLong Gold antifade, Molecular Probes). The samples were allowed to cure for 24-48 h.

Zeiss Elyra PS1

Luxendo MuVi SPIM light-sheet microscope

DeltaVision OMX V3 Blaze system (GE Healthcare) equipped with a 60x/1.42 NA PlanApo oil immersion objective (Olympus), pco.edge 5.5 sCMOS cameras (PCO) and 405, 488, 593 and 640 nm lasers

Two and three days after surgery, sparse labeling of OPCs was achieved by i.p. injection of Tamoxifen (Tam; 180mg/kg bodyweight). Imaging fields of view containing identified OPCs were selected to obtain 10-20 SPOTs per mouse. Chronic 2-photon imaging was performed starting 3 days after surgery. All SPOTs were checked on a daily basis and no z-stack was acquired if no changes occurred. The imaging was performed on custom-built 2-photon microscope (Sutter Instrument Movable Objective Microscope type) using a long-working distance objective (water immersion, 16x magnification, 0.8NA, Nikon) and equipped with a ytterbium-doped laser system at 1045nm and 200fs (Femtotrain, High-Q lasers) or a fiber oscillator 45 laser at 1070nm (Fidelity-2, Coherent) to excite tdTomato labeled cells in the CC. Emission light was detected using a photomultiplier tube (Hamamatsu) after passing a red emission filter (610/75 nm; AHF).

Embryos were imaged on a Luxendo MuVi SPIM light-sheet microscope, using 30x magnification setting on the Nikon 10x/0.3 water objective. The 488 nm laser was used to image nuclei (His-GFP), and the 561 nm laser was used to image transcriptional dots (MCP-mCherry), both at 5% laser power. Exposure time for the green channel was 55 ms and exposure for the red channel was 70 ms. The line illumination tool was used to improve background levels and was set to 40 pixels.

Spherical aberration was minimized using immersion oil with refractive index (RI) 1.514. 3D image stacks were acquired over the whole nuclear volume in z and with 15 raw images per plane (five phases, three angles). The raw data were computationally reconstructed with SoftWoRx 6.5.2 (GE Healthcare) using channel-specific OTFs recorded using immersion oil with RI 1.512, and Wiener filter settings between 0.002-0.006 to generate 3D stacks of 115 nm (488 nm) or 130 nm (593 nm) lateral and approximately 350 nm axial resolution. Multi-channel acquisitions were aligned in 3D using Chromagnon software (Matsuda et al, 2018) based on 3D-SIM acquisitions of multi-colour EdU-labelled C127 cells(Kraus et al, 2017).

bright-field microscopy

spinning disk confocal microscopy

high-voltage electron microscopy (HVEM)

Biological Imaging Methods Ontology (FBbi)

http://purl.obolibrary.org/obo/FBbi_00000243

http://purl.obolibrary.org/obo/FBbi_00000253

http://purl.obolibrary.org/obo/FBbi_00000622

Manual overlay

Alignment algorithm

Image segmentation was performed for each 2D slice using a program called ilastik, which utilizes semantic segmentation. 3D object creation from 2D binary images and feature extraction was performed in a program called Arivis.

Each 3D-SIM image contained one nucleus (in a small number of cases multiple nuclei were present, which did not affect the analysis). The image analysis pipeline contained six main steps: bivalent skeleton tracing, trace fluorescence intensity quantification, HEI10 peak detection, HEI10 foci identification, HEI10 foci intensity quantification, and total bivalent intensity quantification. Note that the normalization steps used for foci identification differ from those used for foci intensity quantification; the former was intended to robustly identify foci from noisy traces, whilst the latter was used to carefully quantify foci HEI10 levels.

Images were deconvolved using the default conservative deconvolution method using DeltaVision Softworx software. Image quantification was carried out using Fiji (Schindelin et al, 2012). Deconvolved images were compressed to 2D images displaying the maximum intensity projection for each pixel across z-stacks listed in Table S7 (column “Projected”). Cell and nuclear areas were outlined using thresholding functions on the background TRITC signal and DAPI signal, respectively. Dots corresponding to transcripts were then counted for both nuclear and cytoplasmic areas for each image by applying the “Find Maxima” command with a noise tolerance specified in Table S7 (column “Maxima”). Bar charts show the mean number of dots per nuclear area and cytoplasmic area across all images for all combined replicates.