Barcode construction and fusion with genetic variant libraries
- Construction of the barcode library
1.1. Mix all of the barcode construction oligonucleotides listed in Table 1 in water to a final concentration of 100 nM each.
1.2. Mix 16 µL of the above oligonucleotide mixture, 2 µL T4 ligase buffer, 2 µL PNK in a PCR tube.
1.3. Incubate this mixture at 37 °C for 30 minutes to phosphorylate any oligonucleotides that may have lost the phosphorylation provided during oligonucleotide synthesis. Incubate at 65 °C for 20 minutes to heat inactive the PNK.
1.4. Add 1 µL T4 ligase (NEB, M0202S) to this mixture and incubate this reaction for 1 hour at room temperature to ligate the oligonucleotides forming the DNA template for the RNA barcodes.
1.5. To amplify this library of DNA templates, dilute the ligation product 10 fold into water and mix 5 µL of this dilution with 25 µL Phusion 2X master mix, 2.5 µL 20X EvaGreen, the barcode amplification primers to a final concentration each of 0.5 µM, and water to a total volume of 50 µL.
1.6. PCR amplify this reaction via limited cycle PCR.
1.7. Gel purify this PCR product using the Zymoclean DNA recovery kit and the manufacturer’s instructions. Elute the product into 20 µL water.
1.8. To create the desired backbone vector for barcode expression, PCR amplify 50 µL of the specific backbone vector and gel purify (as in 1.5-1.7). Elute in 20 µL water.
1.9. Mix 5 µL of the gel purified barcode ligation product, 5 µL of the gel purified barcode expression backbone, and 10 µL HiFi DNA assembly master mix. Incubate for 1 hour at 50 °C to fuse the barcode DNA template with the plasmid backbone, purify using the Zymo DNA clean and concentrator kit according to the manufacturer’s instructions, and elute the plasmid library with 6 µL water.
1.10. To transform this plasmid library into E. coli, mix the assembled plasmids with 10 µL of electrocompetent E. coli on ice, transfer into an electroporation cuvette on ice, and electroporate using an Amaxa Nucleofector II set to bacteria program 3.
1.11. Immediately add 1 mL SOC, transfer into a 14mL culture tube, and incubate at 37 °C on a shaker for one hour.
1.12. Dilute this culture into 50 mL of LB supplemented with 0.1 mg/mL carbenicillin in a sterile, beveled 250 mL flask. Place on the shaker at 37 °C overnight to allow the cells which have taken up a plasmid to grow.
1.13. Miniprep the overnight culture using the Zyppy plasmid miniprep kit according to the manufacturer’s instructions. Elute the final barcode plasmid library into 30 µL Zymo elution buffer and store at -20 °C until needed.
- Merging the barcode expression library with an existing genetic variant library
2.1. Amplify and gel purify the corresponding halves of the barcode plasmid library and an existing genetic variant library by limited-cycle PCR using the appropriate barcode-genetic-variant-fusion primers (as in 1.5 – 1.7).
2.2. Mix 5 µL of each of the purified plasmid library fragments with 10 µL HiFi DNA assembly master mix. Incubate for 1 hour at 50 °C to assemble these PCR fragments, and purify the resulting plasmids using the Zymo DNA clean and concentrator kit according to the manufacturer’s instructions. Elute the plasmid library into 6 µL of water.
2.3. Transform this library into electrocompetent E. coli and then re-isolate this plasmid library via plasmid extraction as described in 1.10 – 1.12.
2.4. To ensure that each E. coli contains at most one plasmid, dilute this plasmid library to a defined concentration of 100 pg/µL in water and re-electroporate into E. coli (as in 1.10 – 1.12).
2.5. Measure the OD600 of the overnight culture and dilute this culture to a concentration of ~1000 cells/µL using the conversion of 800 million cells per 1 unit of OD600.
2.6. Inoculate 2mL of LB containing 0.1 mg/mL carbenicillin with a volume of culture containing the number of cells corresponding to the desired library size. (We typically keep a number of cells that is at most 10% of the expected number of unique barcodes in the original barcode library). Incubate at 37 °C overnight on a shaker to grow this culture to saturation.
2.7. Miniprep 600 µL of the overnight culture (as in 1.13) and elute into 30 µL Zymo elution buffer. Store this plasmid library at -20 °C. It will be used as a source for material for next-generation sequencing.
2.8. To archive the E. coli containing this barcoded genetic-variant library for later high-throughput image-based screening, mix the remaining overnight culture with an equal volume of 50% glycerol. Separate this mixture into 100 µL single-use aliquots and store at -80 °C.
- Sequencing the merged genetic variant and barcode expression library to determine which barcode was associated with each genetic variant
3.1. Dilute the barcoded genetic variant plasmid library (prepared in 2.7) 10-fold in water. To extract the desired regions of this plasmid, where each region contains the UMI, amplify 5 µL of this dilution via limited cycle PCR using the Illumina-sequencing-adaptor primers and gel purify this product (as in 1.5-1.7). Elute the product in 200 µL of water. To address complications associated with sequencing of libraries with identical regions within many different molecules, these primers should contain short regions of random nucleotides of variable length. These variable length regions will introduce apparent variability into these common regions during sequencing.
3.2. Because the desired region to be sequenced is typically larger than the read length associated with the sequencing platform used, repeat 3.1 using different primer sets that allow amplification of different portions of the desired region of these plasmids. It is crucial that each amplification product contain the UMI region so that these different fragments can be associated with the same plasmid during analysis.
3.3. To add the adapters necessary for Illumina sequencing, amplify and gel purify 5 µL of this product (as in 1.5-1.7). To limit the degree of amplification, use these primers at a reduced concentration of 0.05 µM. Elute the product into 10 µL water.
3.4. Quantify the concentration of the elution using the Qubit fluorimeter and dilute to 4 nM in water.
3.5. Sequence these molecules following the MiSeq protocol from Illumina.
3.6. Construct the barcode to genetic variant look-up table by grouping sequencing reads by UMI and assigning to each the most frequently occurring barcode and gene variant seen for that UMI.
- Preparing the library for barcode imaging
1.1. Thaw a 100 µL aliquot of the barcoded genetic-variant library (prepared in 2.8) to room temperature and dilute into 2 mL LB supplemented with 0.1 mg/mL carbenicillin. Incubate on the shaker at 37 °C for 3 hours to allow these cells to grow.
1.2. Optional: If the barcode library is under the control of a plasmid utilizing a relaxed origin, e.g. an origin capable of dividing even when translation is inhibited, barcode expression can be enhanced by inhibiting protein translation. Add 20 µL of chloramphenicol to the culture to a final concentration of 34 µg/mL to inhibit growth. Incubate at 37 °C for an additional 3 hours.
1.3. In parallel, coat an imaging coverslip so that E. coli will stick to it by placing the coverslip in a petri dish and covering with 1% PEI in water for 30 minutes. Wash the coverslip once in PBS. Dry the coverslip by removing the PBS.
1.4. To adhere E. coli to the coverslip, dilute the above culture 10-fold into PBS and pour onto the coated coverslip. To force E. coli cells to lie flat on the surface of the coverslip, spin the petri dish in a centrifuge at 100g for 5 minutes.
1.5. Wash the coverslip once with PBS.
- Prepare the hybridization and imaging buffers for measurement of the barcode RNAs
2.1. Prepare aliquots of water, PBS, 2X SSC, and fixation buffer6 (80% v/v methanol and 20% v/v acetone).
2.2. Prepare an imaging buffer7 which will reduce the rate of fluorophore photobleaching during barcode imaging. This buffer is comprised of 50 mM TrisHCl pH 8, 10% w/v glucose, 2 mM Trolox, 0.5 mg/mL glucose oxidase, and 40 µg/mL catalase in 2X SSC.
2.3. Prepare a cleavage buffer to cleave the disulfide bond linking fluorophores to readout probes, allowing these dyes to be rapidly washed away. This buffer is comprised of 50 mM TCEP in 2X SSC.
2.4. Prepare a master hybridization mix consisting of 5% w/v dextran sulfate, 5% w/v ethylene carbonate, 0.05% w/v yeast tRNA, and 0.1% v/v Murine RNase inhibitor in 2X SSC.
2.5. Aliquot 1 mL of this hybridization master mix into 15 mL falcon tubes, making one tube for each round of hybridization. Add to each tube the appropriate set of readout probes (as described in Table 2) to a final concentration each of 10 nM. Each readout probe will determine the presence of a “1” or a “0” in a given bit, and each round can probe the value of multiple bits simultaneously by using multiple readout probes each conjugated to a spectrally distinct fluorescent dye.
2.6. Load all buffers and hybridization mixes into the fluidics system.
- Image the phenotypes and the barcodes
3.1. Assemble the coverslip into the flow chamber following the manufacturer’s instruction.
3.2. Flow PBS into the chamber and place it on the microscope.
3.3. Proceed with the appropriate high-resolution imaging protocol for the desired phenotype measurement.
3.4. After phenotype imaging, fix the cells by washing the flow chamber with water to remove salts (that can precipitate upon contact with methanol and acetone) and then flowing on the fixation buffer. Incubate the sample at room temperature for 30 minutes in this buffer. Wash again with water to remove residual fixation buffer.
3.5. Flow on the hybridization buffer containing the readout probes assigned to the first hybridization round.
3.6. Incubate at room temperature for 30 minutes to allow the readout probes to hybridize to the complementary barcode RNAs.
3.7. Flow the imaging buffer onto the sample.
3.8. Collect an image of each field-of-view with the illumination channels appropriate to the readout probes utilized. For example, for the readout probes listed in Table 2, collect an image of each field-of-view when illuminated with 750-nm, 650-nm, and 560-nm light to image the readout probes conjugated to Alexa750, Cy5, and ATTO565, respectively, in addition to an image with bright-field illumination for image alignment.
3.9. Collect a brightfield image of this field-of-view which will be used during analysis to correct any small offset of the stage observed between imaging rounds
3.10. Repeat 3.8-3.9 for as many fields of view as desired.
3.11. Flow the cleavage buffer onto the sample to extinguish fluorescence from the sample. Incubate for 15 minutes.
3.12. Flow 2XSSC onto the sample to remove residual cleavage buffer.
3.13. Repeat 3.5-3.12 for each round of hybridization. For example, readout of a 21-bit barcode will require 42 distinct readout probes which, with 3 color imaging, can be measured in 14 rounds of hybridization.
3.14. Recommended: The use of integrated microscope and fluid handling software can automate the repetitive process of imaging, fluid handling and flow, and staining of the sample, substantially simplifying this protocol.
- Identify the barcode expressed within each cell
4.1. Correct inhomogeneities in the illumination using a flat-field correction. This correction can be determined by averaging the fluorescence signal observed across many fields of view.
4.2. Align images from each round of imaging by maximizing the cross-correlation of the bright-field images.
4.3. Segment cells by determining closed boundaries after applying the Canny edge detection algorithm.
4.4. For each cell, calculate the mean fluorescence intensity across all constituent pixels in each image.
4.5. To account for differences in dye brightness and staining between hybridization rounds, normalize the intensity observed for each color channel in each round by the mean intensity observed for that color channel and round across all fields of view.
4.6. For each bit, determine the ratio of the fluorescence signal observed in each cell for the readout probe associated with a “0” signal to that associated with a “1” signal. This “0”-to-“1” ratio will be used to determine if the barcode associated with each cell should be assigned a “1” or “0” in a given bit.
4.7. To determine the best bit-calling-threshold for assigning a bit value from the “0”-to-“1” ratio, select ~100 barcodes known to be present in the sample via next-generation sequencing. Determine the frequency with which these barcodes are detected as a function of this bit-calling-threshold for each bit. Select the set of thresholds that maximize this number.
4.8. For each cell, determine the value of the measured barcode using these thresholds and the measured “0”-to-“1” ratios.
4.9. Discard cells with measured barcodes that were revealed by next-generation sequencing to have been assigned to multiple genetic variants
4.10. Recommended: Some cells will appear dim in both the “1” and “0” channels associated with a given bit, potentially leading to incorrect calling of this bit. Thus, to increase the accuracy, cells with a “1” or “0” brightness below a given threshold can be discarded from subsequent analysis.
4.11. Group cells together that are expressing the same barcode to determine the average properties of the measured phenotype.