Formaldehyde

Wells of six-well plate with cells were rinsed three times with PBS, fixed in 4% formaldehyde (Sigma-Aldrich) for 10 min at RT, and then washed three times 5 min with PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at RT and blocked in 1% BSA in PBS/0.1% Tween-20 for 1 h at RT. Cover slips were incubated in a humid chamber with primary antibody for overnight at 4°C diluted in fresh blocking buffer. The antibody solution was washed with PBST (3x, 5 min) and samples were incubated with corresponding secondary antibody solution (Alexa-Fluor conjugated secondaries) for 2 h at RT in the dark. Cells were washed again 3 × 5 min in PBST, stained with 10 ng/ml DAPI in PBS, rinsed 2x in PBS, and then mounted in Prolong Gold mounting media (invitrogen) for subsequent microscopy acquisition.

HeLa cells were plated on cover glasses in 24-well plates and transfected with 200 ng plasmid. The transfection mix was composed of 100 ng of plasmid for the corresponding Cas13 protein and 100 ng of plasmid for the corresponding crRNA, or 200 ng of crRNA plasmid for crRNA-only samples. 48 h after transfection, cells were fixed in 4% formaldehyde (Sigma-Aldrich) in PBS for 10 min and permeabilized in 70% ethanol for 4 h. Cover glasses were incubated in the ATTO-488 probe-containing hybridization buffer (2× SSC, 10% formamide, 10% dextran sulfate, 0.5 mg/mL yeast tRNA, 50 μg/mL BSA, and 10 mM ribonucleoside vanadyl complexes) overnight at 4 °C in a humidified chamber, and then mounted on a SuperFrost Plus adhesion slide (Epredia) with ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Imaging was performed with the LSM900 inverted confocal microscope (ZEISS). For RNA FISH following immunocytochemistry, the fixation step was performed immediately after secondary antibody staining and then the same RNA FISH protocol was used. Signal intensity were quantified using ImageJ. All quantifications were conducted in a blinded and random manner.

Cells were detached with TrypLE, washed with PBS 1X, and collected to a final number of 30 × 10⁶ for each replicate. Cells were resuspended in 45 mL PBS 1X and 3.5 mL of Fixing Solution (50 mM HEPES–KOH pH 7.5, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) and fixed with 1.1% formaldehyde (Sigma-Aldrich #252,549) for 10 min at RT. The fixing reaction was quenched with 125 mM Glycine and cells were washed with PBS 1X at 2000 g for 6 min. Cells were lysed in Lysis Buffer 1 (50 mM HEPES–KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 10% Glycerol, 0.5% IGEPAL, 0.25% Triton X-100, cOmplete Mini EDTA-Free Protease Inhibitors (Roche)), and washed in Lysis Buffer 2 (10 mM Tris–HCl pH 8, 200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, protease inhibitors). Soluble chromatin was sheared by sonication in Sonication Buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% C₂₄H₄₀O₄, 0.1% SDS, cOmplete Mini EDTA-Free Protease Inhibitors (Roche)) in 1 mL Covaris tubes (Covaris #520,081) using a Covaris 220 system to a chromatin average size of 200–250 bp. Soluble chromatin was separated by collecting the supernatant after centrifugation at 18,000 g for 2 min at 4 °C. Sixty μg or 100 μg of chromatin were immunoprecipitated overnight at 4 °C with 10 μg of antibody. Samples were incubated with 50 μL of Dynabeads protein G magnetic beads (Life Technologies), previously blocked with 0.5% BSA for 4 h at 4 °C. Bead-bound chromatin was washed once with 1 mL of Sonication Buffer, twice with 1 mL of Sonication Buffer supplemented with 500 mM NaCl, twice with 1 mL of Sonication Buffer supplemented with 1 M NaCl and once with 1 mL of LiCl Wash Buffer (20 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% C₂₄H₄₀O₄). Beads-bound chromatin was eluted twice for 15 min at 65 °C in Elution Buffer (50 mM Tris–HCl pH 8.0, 10 mM EDTA, 1% SDS) and de-crosslinked overnight at 65 °C. Chromatin was treated with 0.2 mg/mL of RNaseA at 37 °C for 1 h and with 0.2 mg/mL of Proteinase K at 55 °C for 30 min. DNA was isolated by phenol/chloroform extraction followed by ethanol precipitation and resuspended in 30 μL of 0.1 M TE. Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Laboratories #E7645S) following the manufacturer’s protocol. Libraries were size selected by electrophoresis on Nusieve 3:1 agarose gel to isolate fragments from 250 to 460 bp and DNA purified using Monarcharch DNA Gel Extraction Kit Protocol (New England Laboratories #T1020). The libraries’ quality was evaluated using Tape Station DNA HS D1000 Kit on a Tape Station system (Agilent 4150) and single-end sequenced with Illumina NextSeq 500.

The morphology of the epithelium was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Tissue cultures were washed with DPBS three times successively before fixation for 30 min with sodium bi-phosphate buffer (0.2 M, pH 7.2) with 2% formaldehyde (v/v, Sigma-Aldrich, St. Louis, MO, USA) and 2.5% glutaraldehyde (v/v, Sigma-Aldrich). Fixed tissues were sent in DPBS to Tours University (Tours, France) where the cultures were processed and imaged.

All reagents were purchased from commercial sources; Accutase (BD Biosciences), DMEM glutaMAX (without pyruvate) (Gibco), Dulbecco's PBS (Eurobio Scientific), fetal bovine serum (Gibco), cell lysis buffer (10X) (Cell Signaling), gelatin from porcine skin (type A), formaldehyde, glutaraldehyde, digitonin, Triton X-100, goat serum, NH₄Cl, Pipes (all from Sigma-Aldrich), FluorSave (Calbiochem), and 4',6-diamidino-2-phenylindole (Euromedex). Antibodies against the following proteins were used in this study: Rab4 (ThermoFisher, Research Resource Identifier [RRID]: AB_2269382), Rab5 (Cell Signaling; RRID: AB_2300649), EEA1 (ENZO, ALX-210-239-C100), PI3KC2β (BD Transduction Laboratories; RRID: AB_398865), Myosin Heavy Chain (MYH4) (eBioscience; RRID: AB_2572894), HA (Sigma-Aldrich; catalog no.: H3663; RRID: AB_262051), FLAG (Sigma-Aldrich; catalog no.: F3165; RRID: AB_259529), anti-rabbit Alexa 488 (Thermo Fisher Scientific), and antimouse Alexa 488 (Thermo Fisher Scientific). shRNA constructs include mouse Pik3c2b (stock: TRCN0000360889 [Pik3c2b #1], stock: TRCN0000360890 [Pik3c2b #2]). TRC shRNAs were from Sigma-Aldrich (St. Louis, MO); SHC002 (Sigma-Aldrich) was used as a control.