title: C3PO applied to spatial transcriptomics - 1D and 2D gridmode sequence data
design: C3PO applied to spatial transcriptomics - 1D and 2D gridmode sequence data
library_construction_protocol: Dissecting limbs before labelling, mounting, dehydrating and optical clearing the tissue. Harvesting limb buds: Limbs at approximately E11 developmental stage were dissected from wildtype C56BL/6 mouse embryos in cold PBS  Labelling tissue:Tissue was fixed with 4% PFA shaking at room temperature for 1 hour or overnight at 4C on a shaker. Tissue was then permeabilized and endogenous RNAases deactivated by treatment with 0.025% CHAPS, 0.5% Triton with 0.143Molar Beta-mercaptoethanol in PBS overnight at 4C on a shaker. The sample was quickly washed with PBS and transferred to a PCR tube. Samples were then stained with 10-50_M fluorophore conjugated oligonucleotide with 10% DMSO, 0.01% Triton in PBS overnight in the dark at 4C on a shaker. The following day the samples were placed in a thermocycler and oligonucleotides bound using 6 cycles of 95C for 15 seconds and 25C for 1 minute followed by cooling to 15C. Samples were then washed in PBS twice quickly and once for approximately 1-2hours in the dark at room temperature on a shaker before being post fixed in 4% PFA for 2 hours in the dark at room temperature on a shaker before finally being moved to PBS.  Mounting, dehydrating and clearing samples:Samples were quickly washed in milliQ water before being mounted in 1% low melting point agarose dissolved in Milliq water and filtered through a 0.45_m filter. After mounting, samples were dehydrated by washing in 100% methanol once quickly and twice for >4hours in the dark in glass vials at 4C. Samples were then cleared by washing in 1:2 benzyl alcohol:benzyl benzoate (BABB), once quickly and twice >4hours in the dark in glass vials at 4C. Imaging and bleaching gradients of active fluorophore levels into tissue. Imaging and photo-bleaching:Samples were imaged and bleached on an custom-built OPTiSPIM light sheet mesoscope. Briefly, a 50mW 405nm, a 50mW 488nm, and a 50mW 639nm lasers were used to image and bleach the Alexa405, 6FAM and Alexa647 fluorophores respectively. A 5mW 543nm HeNe laser was used to image the staining uniformity control TAMRA fluorophore. 447BP60, 525BP50, 585BP60, and 700BP75 band pass filters were used for detecting the Alexa405, 6FAM, TAMRA and Alexa647 fluorophores respectively. A 2.5_ N Plan air objective (Leica, NPLAN, NA=0.07) was used to illuminate the samples. A 5_ N Plan Epi air objective (Leica, NPLAN EPI, NA=0.12, WD=14 mm) and a 12 bit cooled Hamamatsu ORCA-ER C4742-80 CCD camera were used for detection. A calibration sample was used to calibrate the bleaching, by placing the sample at different positions relative to the light sheet for each laser line and monitoring the reduction in fluorescent signal with time of exposure at 100% laser power (see SM Fig. S1). Bleaching exposure times for different positions of the sample of interest for C3PO analysis are determined through reading off of these calibration graphs (one for each fluorophore/laser line). Tissue rehydration, dissociation to single cells and ratio sorting with FACS. Processing of photo-bleached samples:After bleaching, the sample received one quick wash and two longer washes (>4hours) in methanol at 4C. The sample was then removed from the LMP agarose and rehydrated with one quick wash and two longer washes (>4 hours) in PBS at 4C. For RNA-based experiments the tissue was digested with 10x Tryple Select (A1217701 Thermofisher with 1/1x10^6 Triton) and 1mg/ml collagenase type III (#07422 Stem Cell Technologies)at 37C 1200 RPM for 2 hours. Tissue samples were periodically mechanically agitated with a gilson pipette. Following tissue dissociation, the sample was centrifuged 3 times at 600g for 5 minutes at room temperature (resusended in 1ml PBS between each centrifugation). Finally, cell pellets were resuspended in 0.5-1ml PBS and strained through a 40_m strainer and processed on a FACS sorter (BD FACSAria II).  FACS:To detect Alexa405 fluorophores a 407nm laser with 450BP40 bandpass filter was used on the FACSAria II. To detect 6FAM fluorophores a 488nm laser with 530BP28 bandpass filter and 505LP longpass filter was used on the Fortessa and a 488nm laser with 525BP50 bandpass filter and 505LP longpass filter was used on the FACSAria. To detect TAMRA fluorophores a 561nm laser with 585BP15 or 610BP20 bandpass filter was used on the FACSAria II. To detect Alexa647 fluorophores a 633nm laser with 670BP14 bandpass filter was used on theFACSAria II. A FACS gate for Side scatter area and Forward scatter was used to remove debris (P1). Singlets were then selected with a Forward scatter height versus area gate (P2). A PE-A (with similar fluorescence profile to TAMRA) versus Forward scatter gates was used to select the fluorescent cells and remove any remaining debris (P3).  For grid-mode transcriptomics ratio sorting was performed where PACB (corresponding to Alexa405), FITC-A (corresponding to 6FAM) and APC-A (corresponding to Alexa647) fluorescent channel levels were normalized to the PE-A (corresponding to TAMRA) fluorescent channel level. At most 2 channels can be used simultaneously for ratio sorting on the FACSAria II. RNA extraction and Library preparation protocol from sorted cells. RNA Extraction:Cells were sorted into 45_l RLT buffer (#79216 Qiagen) and 5_l ProteinaseK (#69506 Qiagen Blood and tissue kit). Samples were then heated at 56C for 1 hour shaking at 1200 RPM. 125_l of RNA clean beads (A63987 Beckman Coulter) were added to the sample, vigorously mixed and let stand at room temperature for 5 minutes. Samples were then placed on a magnet for 5 minutes and washed twice with 70% Ethanol for 30 seconds each. Pellets were air dried for 10 minutes on the magnet before being removed. Beads were resuspended in 15_l of nuclease free water and vigorously mixed. Samples were stood at room temperature for 5 minutes and then placed on a magnet. After 5 minutes the RNA was removed from the tubes. Library preparation:Libraries were prepared using the NEBNext single cell/Low input RNA kit (E6420 New England Biolabs) using all RNA resulting from extraction above. All instructions were followed except 1.0x SPRISelect beads (B23318 Beckman Coulter) were used at every step. 21 cycles of amplification performed on cDNA libraries and 15 cycles of amplification for the final library. Concentration of samples was determined by bioanalyzer or Tapestation and samples pooled at 10nMolar.