TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within <strong>14 d</strong>, with image analysis requiring an additional <strong>2 d</strong>.</p>

[BEFORE_START]
<p><strong>REAGENT SETUP</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>0.25% Triton X-100 </strong>Dilute 0.25 ml 10% Triton X-100 in DEPC-treated H2O to a total volume of 10 ml. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>2X SSC </strong>Dilute 20X SSC in H2O and make up to a total volume of 50 mL. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>1X SSC </strong>Dilute 20X SSC in H2O and make up to a total volume of 50 mL. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>5X SASC </strong>Make 0.75 M sodium acetate, 75 mM tri-sodium citrate, pH to 7.5 using acetic acid in H<sub>2</sub>O to a final volume of 50 ml. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>RCA primer hybridization buffer </strong>Dilute 20X SSC2x SASC, 30% formamide in H2O. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Strip Buffer </strong>(80% formamide in H20, 0.01% Triton-X100 in a final volume of 50 ml. Store at RT for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Cleave Solution 2.1, reconstituted </strong>Mix 1 ml Cleave Solution 2.1 Part 1 with 2.75 ml Cleave Solution 2.1 Part 2. Store at 4°C in dark for up to 24 hours.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p><strong>EQUIPMENT SETUP</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Microscope setup—</strong>Configure a 4 channel microscope with appropriate excitation light sources and emission filters: FITC-488 ex, 490–560 nm em; Cy3–561 nm ex, 563–593 nm em; Texas Red-594 nm ex, 597–647 nm em; Cy5–633 nm ex, 637–758 nm em. Suggested microscope objectives are Plan-Apochromat dry 20× NA 0.75, dry 40× NA 0.8 and water immersion 63× NA 1.3.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Software installation—</strong>Verify that Bio-Formats (<a href="http://loci.wisc.edu/software/bio-formats" target="_blank">http://loci.wisc.edu/software/bio-formats</a>) plug-ins are available for Fiji/ImageJ. Download a free academic version of Canopy Python 2.7 (<a href="https://www.enthought.com/downloads" target="_blank">https://www.enthought.com/downloads</a>) in the home directory on the remote host, and follow the installation instruction (<a href="http://docs.enthought.com/canopy/quick-start/install_linux.html" target="_blank">http://docs.enthought.com/canopy/quick-start/install_linux.html</a>). Canopy Python 2.7 is easy to install and has all the required packages for our FISSEQ software. Install the latest version of ggplot2 and data.table packages in RStudio.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>PC setup</strong></p>
<ul>
<li>Access to a high performance computing cluster (remote host)</li>
<li>
<p>Bowtie 1.0 or earlier (<a href="http://bowtie-bio.sourceforge.net" target="_blank">http://bowtie-bio.sourceforge.net</a>) on the remote host</p>
<p><strong>CRITCAL </strong>Bowtie 2.0 or higher does not work with SOLiD sequencing.</p>
</li>
<li>Fiji/ImageJ (<a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a>) on a PC</li>
<li>MATLAB (<a href="http://www.mathworks.com" target="_blank">http://www.mathworks.com</a>) on the remote host</li>
<li>
<p>Python 2.7 (<a href="https://www.enthought.com/products/canopy/" target="_blank">https://www.enthought.com/products/canopy/</a>) on the remote host</p>
<p><strong>CRITCAL </strong>Other versions of Python lack the required modules for running our script.</p>
</li>
<li>R (<a href="http://www.r-project.org" target="_blank">http://www.r-project.org</a>) and RStudio (<a href="http://www.rstudio.com" target="_blank">http://www.rstudio.com</a>) on a PC</li>
<li>Windows PC or Mac with 16GB RAM minimum</li>
<li><strong>Optional: </strong>SVI Huygens 3D deconvolution software (commercial), Bitplane Imaris 3D rendering software (commercial)</li>
</ul>
<p> </p>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>The protocol workflow is as follows: </strong></p>
<p> </p>
<p style="text-align: center;"><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2mfw26.png" width="629" height="402" data-src="https://s3.amazonaws.com/pr-journal/r2kfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.09.50 PM.png" /></strong></p>
<p style="text-align: center;"> </p>
<p><strong>Timing</strong></p>
<p> </p>
<p><strong>Steps 1–21: </strong>FISSEQ library construction: <strong>2 d</strong></p>
<p><strong>Steps 22–33: </strong>Sequencing and imaging: <strong>10 d</strong></p>
<p><strong>Steps 34–36: </strong>Image pre-processing: <strong>6 h</strong></p>
<p><strong>Steps 37–49: </strong>Image analysis: <strong>12 h</strong></p>
<p><strong>Steps 50–51: </strong>Data analysis: <strong>2 d</strong></p>
<p> </p>
<p> </p>
<p><strong>RT, RCA and SEQUENCING PRIMERS</strong></p>
<p> </p>
<ul>
<li>Random hexamer RT primer, 100 uM in nuclease-free H2O (/5phos/TCTCGGGAACGCTGAAGANNNNNN; hand-mixed, IDT)</li>
<li>RCA primer, 100 uM (TCTTCAGCGTTCCCGA*G*A; * is phosphothioate)</li>
<li>Sequencing primer N : /5phos/TCTCGGGAACGCTGAAGA (HPLC-purified)</li>
<li>Sequencing primer N-1 : /5phos/CTCGGGAACGCTGAAGA (HPLC-purified)</li>
<li>Sequencing primer N-2 : /5phos/TCGGGAACGCTGAAGA (HPLC-purified)</li>
<li>Sequencing primer N-3 : /5phos/CGGGAACGCTGAAGA (HPLC-purified)</li>
<li>Sequencing primer N-4 : /5phos/GGGAACGCTGAAGA (HPLC-purified)</li>
</ul>
<p> </p>
<p> </p>
<p><strong>CONTROL PRIMERS</strong></p>
<p> </p>
<ul>
<li>Adapter-specific probes, 100 uM (/56-FAM/TCTCGGGAACGCTGAAGA)</li>
<li>Adapter-specific probes, 100 uM (/5TYE563/TCTCGGGAACGCTGAAGA)</li>
<li>Adapter-specific probes, 100 uM (/5TEX615/TCTCGGGAACGCTGAAGA)</li>
<li>Adapter-specific probes, 100 uM (/5TYE665TCTCGGGAACGCTGAAGA)</li>
<li>18S rRNA detection primer 1: /5biotin/GCTACTGGCAGGATCAACCAGGTA</li>
<li>18S rRNA detection primer2: /5biotin/TACGCTATTGGAGCTGGAATTACC</li>
<li>18S rRNA detection primer3: /5biotin/GTTGAGTCAAATTAAGCCGCAGGC</li>
<li>18S rRNA detection primer4: /5biotin/TTGCAATCCCCGATCCCCATCACG</li>
<li>28S rRNA detection primer1: /5biotin/CCACGTCTGATCTGAGGTCGCG</li>
<li>28S rRNA detection primer2: /5biotin/CACGCCCTCTTGAACTCTCTCTTC</li>
<li>28S rRNA detection primer3: /5biotin/CTCCACCAGAGTTTCCTCTGGCT</li>
<li>28S rRNA detection primer4: /5biotin/TGAGTTGTTACACACTCCTTAGCG</li>
<li>28S rRNA detection primer5: /5biotin/CGACCCAGCCCTTAGAGCCAATC</li>
<li>28S rRNA detection primer6: /5biotin/GACAGTGGGAATCTCGTTCATCCA</li>
<li>28S rRNA detection primer7: /5biotin/GCACATACACCAAATGTCTGAACC</li>
</ul>
<p> </p>
<p> </p>
<p><strong>EQUIPMENT</strong></p>
<p> </p>
<ul>
<li>4°C and −20°C storage units</li>
<li>Centrifuge for 1.5- and 2-ml tubes</li>
<li>Dry block heater for microtubes at 80°C</li>
<li>Falcon conical centrifuge tubes (15 ml and 50 ml) (Fisher Scientific, cat. nos. 14-959-49B and 14-432-22)</li>
<li>
<p>Flexible plastic I.V. catheter for reagent aspiration (Terumo, part no. SR*FF2419)</p>
</li>
<li>
<p>FocalCheck Fluorescence Microscope Test Slide (Life Technologies, part no. F36909)</p>
</li>
<li>
<p>Glass bottom Mattek dish (Poly lysine-treated: part no. P35GC-1.5-14-C, Poly lysine-treated 96-well plate: part no. P96GC-1.5-5-F)</p>
</li>
<li>
<p>Glass Pasteur pipettes (autoclaved)</p>
</li>
<li>
<p>Incubators at 30°C, 37°C (humidified) and 60°C</p>
</li>
<li>
<p>Inverted confocal microscope, PC and image acquisition software</p>
</li>
<li>
<p>Microscope stage insert, metal (for securely gluing the specimen holder)</p>
</li>
<li>
<p>Non-sterile syringes, 10 ml (BD Biosciences, part no. 301029)</p>
</li>
<li>
<p>RNase-free microtubes (Eppendorf, part no. 0030 121.589)</p>
</li>
<li>
<p>Sealable plastic Tupperware container or Ziploc bags (for CircLigase reaction at 60°C)</p>
</li>
<li>
<p>Vacuum flask, trap, and tubing</p>
</li>
</ul>
<p> </p>
<p><strong>Overview of the procedure</strong></p>
<p> </p>
<p style="text-align: justify;">We begin by fixing cells on a glass slide and performing reverse transcription (RT) in situ. Following RT we degrade the residual RNA to prevent it from competitively inhibiting CircLigase, and cDNA fragments are circularized at 60°C. To prevent cDNA fragments from diffusing away, we incorporate primary amines in cDNA fragments during RT via aminoallyl dUTP, and we then cross-link the primary amines using BS(PEG)9. Each cDNA circle is linearly amplified using RCA into a single molecule containing multiple copies of the original cDNA sequence, and the amine-modified RCA amplicons are cross-linked to create a highly porous and three-dimensional nucleic acid matrix inside the cell (Fig. 1a).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2pfw26.png" width="409" height="583" data-src="https://s3.amazonaws.com/pr-journal/r2pfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.07.44 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In SOLiD sequencing-by-ligation, critical enzymatic steps can be performed directly on a standard microscope at room temperature. First, a sequencing primer is hybridized to multiple copies of the adapter sequence in RCA amplicons, followed by ligation of dinucleotide-specific fluorescent oligonucleotides. After imaging, the fluorophores are cleaved from the ligation complex, and ligation of fluorescent oligonucleotides is repeated six more times to interrogate dinucleotide pairs at every fifth position (Fig.1 b). To fill in the gaps between dinucleotide pairs, the whole ligation complex is stripped off, and four additional sequencing primers with a single base offset are used to repeat dinucleotide interrogation starting from positions n-1, n-2, n-3 and n-4, generating up to 35 raw 3D image stacks representing dinucleotide compositions at all base positions over time.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The raw images are enhanced using standard 3D deconvolution techniques to reduce the background noise, and our MATLAB script performs image alignment to produce TIFF images that are then used for base calling using a separate Python script. The base calls from individual pixels are then aligned to the reference transcriptome using Bowtie, and neighboring pixels with highly similar sequences are grouped into a single object generating a consensus sequence. The final dataset includes the number of individual pixels per object, gene ID, consensus sequence, x and y centroid positions, number of mismatches, base call quality and alignment quality.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">One of the key considerations early in the development of FISSEQ was imaging. Biological patterns, including RNA localization, occur in a scale-dependent manner, in which some patterns are visible at one scale but disappear at another. Therefore, we developed our sequencing method specifically for confocal microscopy using a wide range of objectives, magnification, numerical apertures, scanning speed and depth. Also, autofluorescence, cell debris, and background noise are common in cell imaging, unlike in standard Next-generation sequencing. So we developed an approach to classify individual pixels based on their specific color transitions to detect true signals even in the noisy and/or low intensity environment. Finally, we also developed a way to control the imaging density of single molecules, enabling one to sequence the sequence a large number of molecules in single cells regardless of the microscopy resolution.</p>
<p> </p>
<p> </p>
<p><strong>Applications</strong></p>
<p> </p>
<p style="text-align: justify;">The current FISSEQ protocol is suitable for most cultured cells and tissue sections, including formalin-fixed and paraffin-embedded (FFPE) tissue sections. Whole mount <em>Drosophila </em>embryos, iPS-cell derived embryo bodies (EBs) and organoids are also compatible (Table 1). In FISSEQ each sequencing read has a spatial coordinate, and the reads are binned according to the cellular morphology, sub-cellular location, protein localization, or GFP fluorescence. A statistical test is then applied to identify enriched genes and pathways de novo and discover possible biomarkers of the cellular phenotype<sup>23</sup>. This approach may be combined with padlock probes to detect evolving mutations and RNA biomarkers in cancers<sup>12,13</sup> or to compare gene expression in asymmetric cells or tissues.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2tfw26.png" width="530" height="270" data-src="https://s3.amazonaws.com/pr-journal/r2tfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.10.20 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">FISSEQ may also sequence molecular barcodes in individual cells and transcripts, where expression or reporter (i.e. cDNA, promoter-GFP) libraries are examined in a pool of single cells for massively parallel functional assays and cell lineage tracing. In essence a practically unlimited number of DNA-associated cellular features may now be imaged, enumerated and analyzed across multiple spatial scales using the DNA sequence as a temporal barcode.</p>
<p style="text-align: justify;"> </p>
<p> </p>
<p><strong>Limitations</strong></p>
<p> </p>
<p style="text-align: justify;">On a practical level, equipping a microscope for four-color imaging can cost up to $20,000 for a new filter set and a laser. Most users will need to reserve the microscope for 2–3 weeks so that sequencing can proceed uninterrupted. We have used laser scanning confocal, wide-field epifluorescence and spinning disk confocal microscopes and obtained comparable sequencing data that differ mainly in the read density. With the laser scanning confocal microscope, imaging can take over 30 minutes per stack, but wide-field or spinning disk confocal microscopes can image the same volume in 1–2 minutes. Reagent exchanges are done manually in the current protocol, but FISSEQ samples can remain on the microscope and be sequenced over 2–3 weeks.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">On a technical level, a major limitation of our current protocol is the lack of ribosomal RNA depletion. Initially we used ribosomal RNA as an internal control for library construction, sequencing and bioinformatics; however, this reduced the number of mRNA reads per cell. In primary fibroblasts the ribosomal RNA reads comprised 40–80% of the total<sup>23</sup>; therefore, if one were to deplete the ribosomal RNA24, it may be possible to increase the number of mRNA reads per cell by ~5 fold.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Another limitation is the lack of information on biases in our method. FISSEQ enriches for biologically active genes, enabling discrimination of cell type-specific processes with a small number of reads<sup>23</sup>; however, it is not clear how such enrichment occurs. We hypothesize that active RNA molecules are more accessible to FISSEQ, whereas RNA molecules involved in ribosome biogenesis, RNA splicing, or heat-shock response are trapped in ribonucleoproteins, spliceosomes, or stress granules. It is now important to investigate and understand the molecular basis of such enrichment across multiple cell types and conditions and correlate the result with the observed cellular phenotype.</p>
<p style="text-align: justify;"> </p>
<p> </p>
<p><strong>Experimental design</strong></p>
<p> </p>
<p style="text-align: justify;"><strong>General considerations—</strong>This protocol details the method described in our original report<sup>23</sup>, where endogenous RNAs in cultured fibroblasts were sequenced on a confocal microscope. The availability of a microscope and computational resources will guide the general experimental approach (Table 2). We provide basic computational tools along with a sample dataset, but a background in python, MATLAB, ImageJ and/or R is helpful for analyzing a large number of images. If such expertise is not available, we recommend focusing on a few regions-of-interest with well-demarcated features for comparing gene expression using our custom scripts<sup>23</sup>. After outlining the experiment, one should download our sample image, software and dataset and become familiar with image and data analysis (<a href="http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/" target="_blank">http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/</a>). One should then finalize the experimental design and define the imaging parameters (i.e. area, thickness, resolution, magnification).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2vfw26.png" width="620" height="196" data-src="https://s3.amazonaws.com/pr-journal/r2ufw26.png" data-ofn="Screen Shot 2018-01-06 at 8.10.36 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Cell and tissue fixation—</strong>We have been able to fix and generate in situ sequencing libraries in a wide number of biological specimens (Table 1). The only case in which we failed was a hard piece of bone marrow embedded in Matrigel, which detached from the glass surface after several wash steps. Fixation artifacts can include changes in sub-cellular RNA localization, cell swelling, incomplete permeabilization and RNA leakage. Certain primary cell types are also sensitive to cold<sup>42</sup>, while transformed cell lines or stem cells appear to be less sensitive (Fig. S1). If using FISSEQ to study sub-cellular localization, we recommend fixing cells by adding warm formalin directly into the growth media to a final concentration of 10%.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Cell and tissue sample mounting—</strong>For high resolution imaging we recommend poly lysine- or Matrigel-coated glass bottom dishes, but 96-well plastic bottom plates can be used for simple protocol optimization. Tissue sections can be mounted using a standard mounting procedure, and we advise inexperienced users to consult those practiced in the art of tissue mounting. For non-adherent cell types and whole mount specimens, we recommend fixing samples embedded in Matrigel using 4% paraformaldehyde on a glass bottom dish.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Reverse transcription (RT) in situ—</strong>The length of RT primers should be less than 25 bases to prevent self-circularization. We perform RT overnight for most samples, but one hour is often sufficient for cell monolayers. A negative control without RT should be included to rule out self-circularization of the primer. A positive control primer with the adapter sequence plus a synthetic sequence (~30 additional bases) can be used to check rolling circle amplification (RCA) and imaging parameters. Other than the 5’ region of highly abundant mCherry transcripts<sup>23</sup>, we have not had consistent results with targeted RT. We typically see very few amplicons regardless of the primer design, while random hexamers (24 bases) and poly dT primers (33 bases) work well across all conditions. Some of the possible reasons for failure may include poor target accessibility and competitive inhibition of CircLigase by non-specifically bound sequence-specific RT primers capable of self-circularization. Possible solutions include using LNA-based RT primers for high temperature hybridization<sup>13</sup>, ligation of the adapter sequence post-RT and tiling multiple RT probes across a gene target. We have yet to try these alternatives.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Generation of amplicon matrix—</strong>Aminoallyl dUTP is a dTTP analog commonly used in fluorescence labeling of cDNA<sup>43</sup>, which we utilize for cross-linking nucleic acids; however, the efficiency of RT and RCA is inversely correlated with the concentration of aminoallyl dUTP<sup>23</sup>. The cross-linker, bis(succinimidyl)-nona-(ethylene glycol) or BS(PEG)9, is functionalized with NHS ester groups at both ends<sup>44</sup>, and it forms a stable covalent bond with primary amine groups provided by aminoallyl dUTP at pH 7–9. The cross-linking density can be enhanced by increasing the concentration of aminoallyl dUTP or BS(PEG)9, or by increasing the pH. Cross-linking after RT is optional, but cross-linking of RCA amplicons is essential for high quality sequencing reads.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Sequencing—</strong>We use sequencing-by-ligation<sup>18, 19, 45</sup> (SOLiD<sup>46, 47</sup>) because it works well at room temperature and so a heated stage is not required. SOLiD uses a dinucleotide detection scheme where a base position is interrogated twice per sequencing run<sup>46, 47</sup>, and this can reduce the base calling error rate; however, converting the color sequence to the base sequence is not straightforward due to its propensity to propagate errors, and sequence analysis must remain in the color space (Box 1). In comparison, sequencing-by-synthesis (Illumina) works at 65°C for primer extension and cleavage and utilizes proprietary fluorophores, requiring a heated flow-cell and a custom imaging set-up. Since sequencing-by-synthesis can generally yield a much longer read length, we are currently investigating its compatibility with FISSEQ.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2nfw26.png" width="559" height="436" data-src="https://s3.amazonaws.com/pr-journal/r2nfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.06.39 PM.png" /></p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2rfw26.png" width="568" height="508" data-src="https://s3.amazonaws.com/pr-journal/r2rfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.08.43 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Partition sequencing—</strong>T4 DNA ligase has a single-base specificity at the ligation junction<sup>18</sup>, and sequencing primers differing by one base can recognize different sets of amplicons<sup>23</sup>. By dividing imaging over multiple separate runs, spatially overlapping amplicons can be enumerated using multiple sequencing primers even on a low resolution microscope; however, this requires full automation for the increased number of sequencing runs per sample. Without automation partition sequencing is better suited for quantifying short barcode sequences rather than full RNA sequences in situ<sup>12</sup> (Fig. 3).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2qfw26.png" width="379" height="575" data-src="https://s3.amazonaws.com/pr-journal/r2qfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.08.30 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Imaging—</strong>Epifluorescence microscopy can generate a reasonable number of alignable reads from relatively thin specimens (&lt;5-um), such as HeLa cells<sup>23</sup>, but thicker samples require confocal microscopy to obtain high density reads. Spinning disk confocal microscopy is significantly faster than laser scanning confocal microscopy, and it has a good balance of imaging speed and axial resolution. An automated stage capable of finding a z-stack across multiple x-y tiles is highly desirable (Table 2).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In FISSEQ individual amplicons can be detected using objectives with N.A. 0.4 or greater. The magnification required is determined by the biological question and the amplicon density<sup>48</sup>. Typically we use a 20× N.A. 0.75 objective to examine tissue sections and cultured cell mono-layers, while 40× N.A. 0.8 and 63× N.A. 1.2 water immersion objectives are used for high-resolution imaging of single cells. We have observed noticeable chromatic aberration in our experiments, depending on the objectives used. The degree of chromatic aberration should be measured using image calibration beads (i.e. FocalCheck Fluorescence Microscope Test Slide) prior to sequencing and calibrated by the microscope vendor if necessary.</p>
<p> </p>
<p style="text-align: justify;">For each imaging set-up the user should determine the ideal Nyquist rate. This value can be calculated using <a href="http://www.svi.nl/NyquistCalculator" target="_blank">http://www.svi.nl/NyquistCalculator</a>. The x-y pixel and z-step sizes should not be greater than 1.7 times the Nyquist value for image deconvolution. Four color imaging should proceed from the longest to shortest wavelength (i.e. Cy5, Texas Red, Cy3, FAM), and an intensity histogram should be used to adjust laser power to prevent saturated pixels. The intensity histogram should be consistent across fluorescence channels and sequencing cycles. To use our software the image file name must be standardized: &lt;Position&gt;_&lt;Primer #&gt;_ &lt;Ligation #&gt;_&lt;Date_Time&gt;.extension (e.g. 06_N1_2_2013_10_25_11_57_18.czi).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Image analysis tools—</strong>In practice the extent of image processing and analysis is dictated by the available imaging tools and computing resources<sup>49</sup>. We use Bitplane Imaris for data visualization and movie creation and SVI Huygens for 3D deconvolution. While they are easy to use, scalable and relatively fast, their cost may be out of reach for small labs; however, free and/or open source alternatives are also available<sup>49–51</sup>.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Image deconvolution—</strong>We use 3D deconvolution<sup>52</sup> to reduce the out-of-focus background and improve the quality of base calls (Fig. 5a). High quality 3D deconvolution requires sampling near the Nyquist rate, but this increases the image acquisition and deconvolution time as well as the file size. We generally recommend using high-quality confocal imaging and minimal 3D deconvolution for FISSEQ. Using 3D deconvolution to compensate for low quality imaging will not necessarily improve the quality or number of sequencing reads. We provide a sample dataset containing raw and deconvolved image stacks from a successful 30-base sequencing experiment for practice (<a href="http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/" target="_blank">http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/</a>).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r2sfw26.png" width="494" height="696" data-src="https://s3.amazonaws.com/pr-journal/r2sfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.08.59 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Image registration, base calling and sequence alignment—</strong>As long as the input image files are correctly named, our software will generate the maximum intensity projection, register images and correct for chromatic shifts<sup>23</sup> (Fig. 5b). The resulting images are used for base calling and sequence alignment to human RefSeq (Fig. 5c), but our software does not generate z-coordinates for sequencing reads, as it uses maximum intensity projection for base calling. We provide a sample data output and screen logs for troubleshooting our bioinformatics software (<a href="http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/" target="_blank">http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/</a>).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Data analysis—</strong>Our software generates a tab delimited text file containing 10,000 to 50,000 aligned reads per field of view. We recommend RStudio with the latest version of R installed for plotting reads by RNA classes, position, cluster size, quality, gene name, strand, etc. We provide a sample R session file used for FISSEQ data analysis as an introduction to statistical computing and assessing the quality of FISSEQ dataset (<a href="http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/" target="_blank">http://arep.med.harvard.edu/FISSEQ_Nature_Protocols_2014/</a>).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Level of expertise required for the protocol—</strong>FISSEQ is at the interaction of cell imaging and functional genomics, and it has generated much interest from cell biologists not familiar with RNA-seq. Our protocol is aimed at such researchers, who are familiar with cell image analysis but have few computing skills (Fig. 6). FISSEQ library construction can be performed by anyone with basic molecular biology skills, but image acquisition is best done with help from an imaging core specialist for the initial set-up. Once the equipment, software, imaging and deconvolution parameters are finalized, a capable technician, graduate student, or post-doc can perform manual sequencing on a microscope with some training and practice. Image and sequence analysis using our software can be performed by anyone familiar with the Unix environment, but statistical data analysis requires either a graduate student or post-doc familiar with statistical tools and concepts.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Considerations about the laboratory facilities—</strong>All steps in FISSEQ library construction can be carried out in a standard laboratory setting. A vacuum line facilitates solution aspiration and reagent exchanges, and we do not find RNA degradation or PCR contamination to be a significant problem in our method. We advise having a dedicated microscope with proper excitation and emission filters on a vibration isolation table in a low traffic area.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>TROUBLESHOOTING </strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong><img src="https://s3.amazonaws.com/pr-journal/r2jfw26.png" width="779" height="727" data-src="https://s3.amazonaws.com/pr-journal/r2jfw26.png" data-ofn="Screen Shot 2018-01-06 at 8.10.56 PM.png" /></strong></p>
<p style="text-align: justify;"> </p>
<p> </p>
<p><strong>Supplementary Material</strong></p>
<p> </p>
<p>Refer to Web version on PubMed Central for supplementary material.</p>
<p> </p>
<p><strong>Acknowledgments</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Funded by NIH CEGS grant P50 HG005550. J.H.L. and coworkers funded by NHBLI grant RC2HL102815, Allen Institute for Brain Science, and NIMH grant MH098977. E.R.D. funded by NIH grant GM080177 and NSF GRF grant DGE1144152.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>For additional information and references please see the attached full manuscript. </strong></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.