TITLE: Single Cell Calling Cards Library Preparation
AUTHORS: Arnav Moudgil, Robi D. Mitra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to create calling card libraries from single cell RNA. We assume you have successfully transformed cells with </span><span style = "font-style:italic;">piggyBac</span><span> self-reporting transposons and either undirected </span><span style = "font-style:italic;">piggyBac</span><span> transposase or your favorite transcription factor (YFTF) fused to </span><span style = "font-style:italic;">piggyBac</span><span>. We also assume you have optimized the dissociation protocol for your specific cells or tissues and can generate single cell suspensions.</span></div></div>

[STEPS]
?. [Single Cell Barcoding and Reverse Transcription]
Prepare cells for isolation and encapsulation in gel bead emulsions (GEMs). If your experiment involves a piggyBac transposase with PB-SRT-Puro transposons, cells that have survived sleection should be dissociated and resuspended in solution. If you are using piggyBac with PB-SRT-tdTomato, we recommend using FACS to isolate tdTomato-positive cells, running cells transfected with PB-SRT-tdTomato alone as a gating control.
?. [Single Cell Barcoding and Reverse Transcription]
Follow 10x's instructions for GEM Generation & Barcoding, with this modification:Proceed with Steps 1.2–1.5 as instructed: loading the Single Cell 3' chip, running the controller,  transferring GEMs, and reverse transcription.Incubate the RT reaction under standard conditions.Set lid temperature to Hold at
?. [Single Cell Barcoding and Reverse Transcription]
Step 2.1: clean the GEM-RT mixture using the Recovery Agent and DynaBeads MyOne Silane per 10x's instructions. At the final elution stage, add 36.5 µl Elution Solution I to the tube, mix by pipetting, and incubate at room temperature for 1 minute. Place the tube in a 10x Magnetic Separator in the Low position until the solution turns clear. Transfer 36 µl of the eluted sample to a new tube.
?. [Single Cell Barcoding and Reverse Transcription]
Divide the eluate into two 18-µl aliquots. These can be stored at –20ºC until needed. One aliquot will be used for scRNA-seq library preparation, while the other will be used to generate scCC libraries.
?. [Single Cell RNA-seq Library Preparation and Sequencing]
To continue preparing scRNA-seq libraries, we need to add the template switch oligonucleotide to first strand synthesis products from the RT reaction. Take one of the 18 µl aliquots and thaw on ice.
?. [Single Cell RNA-seq Library Preparation and Sequencing]
Prepare the following 1X master mix:20 µl Maxima 5X RT buffer20 µl 20% w/v Ficoll PM-40010 µl 10 mM dNTPs2.5 µl RNaseOUT2.5 µl 100 µM 10x_TSO
?. [Single Cell RNA-seq Library Preparation and Sequencing]
To the mix, add 18 µl of first strand RT product and 22 µl H2O. Add 5 µl Maxima H– RTase to the reaction, flick to the mix, and centrifuge briefly.
?. [Single Cell RNA-seq Library Preparation and Sequencing]
Incubate:
?. [Single Cell RNA-seq Library Preparation and Sequencing]
Clean up following 10x's post GEM-RT Cleanup protocol, starting with the addition of DynaBeads MyOne Silane (Step 2.1, part D). Clean samples per manufacturer's instructions.
?. [Single Cell RNA-seq Library Preparation and Sequencing]
Complete cDNA amplification and library construction according to the 10x's instructions (Steps 2.2–3.7). For each sample, record which index sample index was used for the final PCR. Quantiate each library by running a 1:10 dilution on an Agilent TapeStation High Sensitivity D1000 ScreenTape.
?. [Single Cell RNA-seq Library Preparation and Sequencing]
Finished scRNA-seq libraries can be pooled and sequenced on Illumina MiSeq, NextSeq, HiSeq, and NovaSeq platforms.
?. [Amplification of Self-Reporting Transcripts]
To prepare single cell calling cards libraries, we start by amplifying self-reporting transcripts from the other aliquot of first-strand synthesis product. As before, thaw the remaining 18 µl aliquot on ice.
?. [Amplification of Self-Reporting Transcripts]
Prepare the following PCR mix in PCR tube:25 µl of 2X Kapa HiFi Hotstart Readymix18 µl of first-strand synthesis product6 µl of ddH2O1 µl of PCR primer cocktailKeep on ice until ready for PCR.
?. [Amplification of Self-Reporting Transcripts]
Prepare a PCR primer cocktail in a PCR tube:5 µl of 100 µM Bio_Illumina_Seq1_scCC_10X_3xPT primer5 µl of 100 µM Bio_Long_PB_LTR_3xPT primer10 µl of Low TE BufferMix by vortexing and spin down briefly. This cocktail can be stored at –20ºC.
?. [Amplification of Self-Reporting Transcripts]
Perform PCR using the following thermocycling parameters:98ºC for 3 minutes20 cycles of:    98ºC for 20 seconds    67ºC for 30 seconds    72ºC for 5 minutes72ºC for 10 minutes4ºC forever.The number of cycles may need to be adjusted depending on the cell type and number of cells represented in the library. If uncertain, you can use 9 µl of first-strand synthesis product as template, reserving the other 9 µl for another round of PCR with more cycles as needed.
?. [Purification of PCR Products]
Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.
?. [Purification of PCR Products]
Add 30 µl beads to the 50 µl PCR mixture (0.6x ratio). Mix by pipetting 10 times until evenly dispersed.
?. [Purification of PCR Products]
Incubate at room temperature for
?. [Purification of PCR Products]
Place on a magnetic rack for Aspirate supernatant and discard.
?. [Purification of PCR Products]
While the tube is still on the rack, add 200 µl of 70% ethanol and incubate ≥ 30 seconds. Aspirate supernatant and discard.
?. [Purification of PCR Products]
Repeat Step #20
?. [Purification of PCR Products]
Air dry the pellet at room temperature for
?. [Purification of PCR Products]
Remove the tube from the magnetic rack. Add 40 µl QIAGEN Elution Buffer to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for
?. [Purification of PCR Products]
Place on magnetic rack for  or until supernatant is clear. Transfer supernatant to new 1.7 ml tube.
?. [Purification of PCR Products]
Take 1 µl of the eluate and dilute in 9 µl of ddH2O to make a 1:10 dilution. Quantitate on TapeStation using a High Sensitivity D5000 ScreenTape. Measure the molar concentration of the sample, taking everything from 250 bp to 7000 bp. Ideally, the diluted sample will be at least 750 pM, corresponding to 7.5 nM for the original eluate. If you kept half of the template aside, you can increase the number of PCR extension cycles until you get to a minimum of 7.5 nM of product.This is what a representative TapeStation trace looks like. The library should be smooth and unimodal.
?. [Single Cell Calling Cards – Circularization]
Thaw an aliquot of CB – Circularization Buffer 10X on ice.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Add 9 µl of PS1 – Exonuclease directly to the overnight circularization mixture. Flick to mix, spin down, and incubate as following:
?. [Single Cell Calling Cards – Shearing and Capture]
Transfer the entire sample (now approximately 320 µl) to a Covaris T6 tube. Add ddH2O as necessary to fill to the top, then cap the tube. Check to make sure there are no air bubbles.
?. [Single Cell Calling Cards – Circularization]
Add the following components to a new 1.7 ml tube in this order:300 fmol self-reporting transcripts from Step 25ddH2O up to a total of 268 µl30 µl CB2 µl Circularization LigaseTo calculate what volume of eluate corresponds to 300 fmol, divide 300 by the molar concentration (in nM) of the eluate. For example, if the concentration is 10 nM, 300 fmol/10 nM = 30 µl, and consequently you would need 238 µl ddH2O. If you have less than 300 fmol total, you may proceed but might need to make adjustments at the final PCR step. If you do not have a way to quantitate the molarity of your solution, we have observed that 300 fmol of self-reporting transcripts is (very) approximately 200 ng.Mix by flicking the tube and spin down briefly. Incubate at overnight (12-16 hours).
We strongly encourage you to calculate the volume of SRT solution based on molarity, not by mass. The circularization reaction is sensitive to starting concentration. If overloaded, it can lead to excess intermolecular ligations and, subsequently, increased noise with respect to the assignment of insertions to cell types.
?. [Single Cell Calling Cards – Exonuclease and Setup]
While the exonuclease digestion proceeds, prepare for the rest of the library preparation. Fill a large ice bucket with ice. Thaw, on ice, aliquots of:STL – Stop Ligation BufferERP3 – End Repair MixATL2 – A-tailing MixLIG2 – Ligation MixEPM – Enhanced PCR MixAlso thaw the following oligonucleotides:scCC_P5_adapter (100 µM)scCC_P7_adapter (100 µM)scCC_P5_primer (25 µM)scCC_P7_primer (25 µM)Also thaw the following oligonucleotides:scCC_P5_adapter (100 µM)scCC_P7_adapter (100 µM)scCC_P5_primer (25 µM)scCC_P7_primer (25 µM)Finally, thaw NEBuffer 2
?. [Single Cell Calling Cards – Exonuclease and Setup]
While the exonuclease incubates, anneal the scCC adapters. Prepare the following mixture in a PCR tube, using a different indexed scCC_P7_adapter for each sample:4.5 µl scCC_P5_adapter4.5 µl scCC_P7_adapter1 µl NEBuffer 2
?. [Single Cell Calling Cards – Exonuclease and Setup]
The exonuclease digestion should be complete by now. Add 12 µl STL – Stop Ligation Buffer. Flix to mix and centrifuge gently.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Prepare the streptavidin-coated magnetic beads. These instructions are for 1 sample; up to 5 can be prepared in a single 1.7 ml tube. Resuspend Dynabeads M-280 by vortexing briefly.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Remove from rack and add 300 µl BBB. Beads can be stored at room temperature until needed.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Transfer 20 µl of beads to a clean 1.7 ml tube.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Place on a magnetic rack for 1 minute. Once clear, aspirate and discard supernatant.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Add 40 µl BBB – Bead Bind Buffer. Incubate for 1 minute, then aspirate and discard supernatant.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Repeat Step #35.
?. [Single Cell Calling Cards – Exonuclease and Setup]
Anneal scCC adapters in a thermocycler using the following settings:95ºC for 5 minutes70ºC for 15 minutesRamp down to 25ºC as slowly as possible25ºC for 5 minutes4ºC foreverscCC adapters can be kept on ice until needed.
Adapters should be prepared fresh. NEBuffer 2 contains magnesium salts which can promote DNase activity, leading to degradation of adapters.
?. [Single Cell Calling Cards – Shearing and Capture]
Shear DNA on a Covaris ultrasonicator. Here are recommended settings for various models (we have tested this protocol on the E220):  ABCD1ModelS2S220E2202Peak Power IntensityN/A2402003Intensity8N/AN/A4Duty Cycle/Factor20%20%20%5Cycles Per Burst2002002006Time4040407Temperature666
ABCD1ModelS2S220E2202Peak Power IntensityN/A2402003Intensity8N/AN/A4Duty Cycle/Factor20%20%20%5Cycles Per Burst2002002006Time4040407Temperature666
?. [Single Cell Calling Cards – Shearing and Capture]
Transfer the sample to a new 1.7 ml tube. Add 300 µl of bead solution to the sheared DNA.
?. [Single Cell Calling Cards – Shearing and Capture]
Incubate If incubating on a thermoshaker, shake at 1000 RPM. Otherwise, flick to mix every 2 minutes.
?. [Single Cell Calling Cards – Shearing and Capture]
Centrifuge briefly (5-10 seconds), then place on a magnetic rack for 1 minute. Discard the supernatant.
?. [Single Cell Calling Cards – Shearing and Capture]
Wash 4 times with BWB – Bead Wash Buffer:Add 200 µl BWBRemove from rack, flick to mix, and spin down briefly (1-2 seconds)Place on rack for 30 secondsDiscard supernatantRepeat for a total for 4 washes
?. [Single Cell Calling Cards – Shearing and Capture]
Wash 2 times with RSB – Resuspension Buffer:Add 200 µl RSBRemove from rack, flick to mix, and spin down brieflyPlace on rack for 30 secondsDiscard supernatantFor the second wash, do not discard supernatant until ready to add the master mix in the next step.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Prepare master mixes for End Repair and A-Tailing as follows.1X End Repair Master Mix:40 µl ERP3 – End Repair Mix60 µl ddH2O1X A-Tailing Master Mix:12.5 µl ATL2 – A-Tailing Mix17.5 µl ddH2O1X A-Tailing Master Mix:12.5 µl ATL2 – A-Tailing Mix17.5 µl ddH2O
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Discard all supernatant from the DNA sample. Centrifuge briefly, then place on a magnetic rack.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Use a 10 µl pipette to aspirate any residual supernatant.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Add 100 µl End Repair reaction mix, remove from the rack, flick to mix, and centrifuge briefly (do not allow beads to pellet).
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Incubate If incubating on a thermoshaker, shake at 1000 RPM, to prevent beads from settling.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Centrifuge briefly (5-10 seconds), then place on a magnetic rack for 1 minute. Discard the supernatant.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Wash 4 times with BWB – Bead Wash Buffer:Add 200 µl BWBRemove from rack, flick to mix, and spin down briefly (1-2 seconds)Place on rack for 30 secondsDiscard supernatantRepeat for a total for 4 washes
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Wash 2 times with RSB – Resuspension Buffer:Add 200 µl RSBRemove from rack, flick to mix, and spin down brieflyPlace on rack for 30 secondsDiscard supernatantFor the second wash, do not discard supernatant until ready to add the master mix in the next step.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Discard all supernatant from the DNA sample. Centrifuge briefly, then place on a magnetic rack. Use a 10 µl pipette to aspirate any residual supernatant.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Add 30 µl A-Tailing reaction mix, remove from the rack, flick to mix, and centrifuge briefly (do not allow beads to pellet).
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Incubate If incubating on a thermoshaker, shake at 1000 RPM, to prevent beads from settling.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Add the following components in order to the A-tailing mix:(30 µl A-tailing reaction)2.5 µl LIG2 – Ligation Mix4 µl ddH2O1 µl annealed scCC adapterFlick to mix and centrifuge briefly (do not allow beads to pellet).
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Incubate
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Add 5 µl STL – Stop Ligation Buffer. Flick to mix.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Centrifuge briefly (5-10 seconds), then place on a magnetic rack for 1 minute. Discard the supernatant.
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Wash 4 times with BWB – Bead Wash Buffer:Add 200 µl BWBRemove from rack, flick to mix, and spin down briefly (1-2 seconds)Place on rack for 30 secondsDiscard supernatantRepeat for a total for 4 washes
?. [Single Cell Calling Cards – End Repair, A-Tailing, and Adapter Ligation]
Wash 2 times with RSB – Resuspension Buffer:Add 200 µl RSBRemove from rack, flick to mix, and spin down brieflyPlace on rack for 30 secondsDiscard supernatantFor the second wash, do not discard supernatant until ready to add the master mix in the next step.
?. [Single Cell Calling Cards – Final PCR and Purification]
Prepare a 1X PCR master mix in a new 1.7 ml tube:20 µl EPM – Enhanced PCR Mix28 µl ddH2O1 µl scCC_P5_primer (25 µM)1 µl scCC_P7_primer (25 µM)
?. [Single Cell Calling Cards – Final PCR and Purification]
Discard all supernatant from the DNA sample. Centrifuge briefly, then place on a magnetic rack. Use a 10 µl pipette to aspirate any residual supernatant.
?. [Single Cell Calling Cards – Final PCR and Purification]
Add 50 µl PCR reaction mix to the sample and pipette to mix. Transfer to PCR tubes.
?. [Single Cell Calling Cards – Final PCR and Purification]
Incubate in a thermocycler with the following settings:98ºC for 30 seconds15 cycles of:    98ºC for 10 seconds    60ºC for 30 seconds    72ºC for 2 minutes72ºC for 5 minutes4ºC forever.
If you started with less than 300 fmol of self-reporting transcripts, you can increase the number of extension cycles here. More cycles will increase the risk of artifacts, however, so we recommend increasing by the minimum necessary to obtain reasonable sequencing libraries. The most we have pushed this PCR is to 17 extension cycles.
?. [Single Cell Calling Cards – Final PCR and Purification]
Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.
?. [Single Cell Calling Cards – Final PCR and Purification]
Place PCR tubes on a magnetic rack for 1 minute. Transfer 50 µl of supernatant to new tubes.
?. [Single Cell Calling Cards – Final PCR and Purification]
Add 35 µl beads to the 50 µl PCR mixture (0.7x ratio). Flick to mix and centrifuge briefly.
?. [Single Cell Calling Cards – Final PCR and Purification]
Incubate at room temperature for
?. [Single Cell Calling Cards – Final PCR and Purification]
Place on a magnetic rack for Aspirate supernatant and discard.
?. [Single Cell Calling Cards – Final PCR and Purification]
Add 200 µl of 70% ethanol and incubate ≥ 30 seconds. Aspirate supernatant and discard.
?. [Single Cell Calling Cards – Final PCR and Purification]
Repeat Step #72
?. [Single Cell Calling Cards – Final PCR and Purification]
Air dry the pellet at room temperature for
?. [Single Cell Calling Cards – Final PCR and Purification]
Remove the tube from the magnetic rack. Add 25 µl RSB – Resuspension Buffer to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for
?. [Single Cell Calling Cards – Final PCR and Purification]
Place on magnetic rack for  or until supernatant is clear. Transfer supernatant to new 1.7 ml tube.
?. [Single Cell Calling Cards – Final PCR and Purification]
Make a 1:10 dilution of the eluate and quantitate on TapeStation using a High Sensitivity D1000 ScreenTape. Measure the molar concentration of the sample, taking everything from 200 bp to 1300 bp.This is what a representative TapeStation trace looks like. The library should be smooth and unimodal, peaking between 500-700 bp. Occasionally, you may see a primer-dimer peak. However, as scCC libraries are sequenced from the middle and not the ends, the primer-dimer product will not sequence on the Illumina flow cell.
?. [Single Cell Calling Cards – Sequencing]
Single cell calling cards libraries use a mix of standard and custom primers for sequencing and rely on dual-indexing for proper demutiplexing. We have sequenced scCC libraries on Illumina NextSeq 500 machines, using v2 Reagent Cartridges. These libraries use the standard Illumina primers BP10 and BP14 for read 1 and index 2, respectively. Read 1 sequences the cell barcode and unique molecular index (UMI), while index 2 reads into the terminal repeat of the piggyBac transposon, confirming that molecules successfully circularized.In addition, we use the custom sequencing primers scCC_PB_CustomRead2 and scCC_CustomIndex1 for read 2 and index 1, respectively. Read 2 anneals at the end of the transposon and sequences into the genome. The first six base pairs typically begin "GGTTAA", which are the terminal two base pairs of the piggyBac repeat followed by the insertion site tetramer. The remainder of the read is genomic DNA sequence. Index 1 sequences the sample-specific sequence on the scCC adapter and is used to demultiplex libraries.
Due to the low complexity nature of calling card libraries, we recommed adding PhiX at a final concentration of 50%.
?. [Single Cell Calling Cards – Sequencing]
While index 1 should be sufficient to demultiplex libraries, we have observed a biphasic response when sequencing scCC libraries at low and high concentrations. At low library concentrations, (e.g., 1-2%) the index 1 read generates high-quality reads and can demultiplex libraries; however at higher concentrations (i.e., 50%) the index 1 read can fail, yielding all N's. If this happens, libraries can be demultiplexed by the index 2 read alone: scCC reads that have successfully circularized will have "GCGTCAAT" as the index 2 sequence.After this, scCC reads can be assigned to specific samples using the cell barcodes obtained from the corresponding scRNA-seq libraries. Different libraries may, by chance, have cells that share the same cell barcode. Typically, these represent a very small fraction of cells (