TITLE: Bulk Calling Cards Library Preparation
AUTHORS: Arnav Moudgil, Michael N. Wilkinson, Xuhua Chen, Robi D. Mitra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to create calling card libraries from bulk RNA. This protocol assumes 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>. Your cells are now ready for RNA extraction, SRT amplification, and library preparation. </span></div></div>

[STEPS]
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Harvest cells. Process each replicate independently. Do not overload gDNA Eliminator columns. If you have more than 107 cells, split cells in half and process on two columns, then merge the RNA pools. Adherent cells may have to be dissociated using trypsin or a cell scraper.Pellet cells by centrifuging at 300g for 5 minutes. Aspirate all of the supernatant.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Add Buffer RLT Plus (with added 2-mercaptoethanol) to the pellet. Use the following table as a guide.  AB1# cellsBuffer RLT Plus2350 µl35e6 to 1e7600 µlMix by vortexing or pipetting.
AB1# cellsBuffer RLT Plus2350 µl35e6 to 1e7600 µl
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Homogenize the lysate by vortexing briefly, then letting rest on bench for 1 minute. Alternatively, cells can be homogenized using QIAshredder spin columns or by repeatedly passing through a 20-gauge needle.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Transfer lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube. Centrifuge for 30 seconds at ≥ 8,000g.  Ensure no liquid remains on the column membrane. Repeat centrifugation if necessary. Keep the flow-through and discard the column.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Add 1 volume (i.e. 350 or 600 µl) 70% ethanol to the flow-through and mix by pipetting.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Transfer up to 700 µl of the sample to an RNEasy spin column placed in a 2 ml collection tube. Spin for 15 seconds at ≥ 8,000g. Discard the flow-through. If sample volume was greater than 700 µl, centrifuge sample in successive batches on the same column, discarding the flow-through at every step.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Add 700 µl of Buffer RW1 to the column and spin for 15 seconds at ≥ 8,000g to wash. Discard flow-through.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Add 500 µl of Buffer RPE to the column. Spin for 15 seconds at ≥ 8,000g. Discard flow-through.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Repeat Step 8 but spin for 2 minutes. Discard the flow-through and the collection tube.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Prepare DNase solution by adding 10 µl of DNaseI to 70 µl of Buffer RDD for each sample. Add 80ul of DNase solution to each column. Incubate at room temperature for 15 minutes.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Add 350 µl of Buffer RW1 to the column and spin for 15 seconds at ≥ 8,000g to wash. Discard flow-through.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Place the spin column in a new collection tube and centrifuge for 1 minute at ≥ 8000g.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Place the spin column in a new 1.5 ml collection tube. Add 40 µl RNase-free water to the column and spin for 1 minute at ≥ 8,000g to elute RNA. RNA can be stored at –80ºC.
?. [RNA Extraction with QIAGEN's RNEasy Plus Mini Kit]
Dilute 1 µl of RNA in 9 µl of ddH2O and quantitate using the Qubit RNA HS Assay Kit.
?. [cDNA Synthesis]
For cDNA synthesis, continue processing each replicate separately. Prepare the reverse transcription (RT) reaction mix:2 µg total RNA1 µl of 50 µM SMART_dT18VN primer1 µl of 10 mM dNTPsRaise to 14 µl with ddH2OIncubate RT mix at for Place on ice for 1 minute
?. [cDNA Synthesis]
Add the following to the RT mix:4 µl 5X Maxima RT Buffer1 µl RNaseOUT1 µl of 0.5X Maxima RT H Minus enzyme (1:1 mixture of 1X Maxima RT Buffer and Maxima RT H Minus enzyme = 100 U)Mix by pipetteing and incubate at for
?. [cDNA Synthesis]
Heat inactivate the reaction by incubating at for
?. [cDNA Synthesis]
Clean up reaction using 1 µl RNase H and incubating at for
Digestion with RNase H removes the complementary RNA strand from the DNA-RNA first strand duplex. This is thought to aid amplification of longer cDNA molecules (> 1 kb)
?. [cDNA Synthesis]
cDNA can be stored at –20ºC
?. [Amplification of Self-Reporting Transcripts]
This PCR will specifically amplify self-reporting transcripts from cDNA libraries. Prepare the following solution:25 µl 2X Kapa HiFi HotStart ReadyMix1 µl of 25 µM Reverse Primer (SMART)2 µl of cDNA21 µl of ddH2O1 µl of Forward Primer, either:    25 µM SRT_PAC_F1 primer, if using PB-SRT-Puro    25 µM SRT_tdTomato_F1, if using PB-SRT-tdTomatoIf you have multiple replicates, amplify them separately.
This PCR can be run as half-size reactions by halving each of the listed volumes. If you find yourself doing this PCR repeatedly, this can be a way to decrease costs.
?. [Amplification of Self-Reporting Transcripts]
Perform PCR using the following thermocycling parameters:95ºC for 3 minutes20 cycles of:    98ºC for 20 seconds    65ºC for 30 seconds    72ºC for 5 minutes72ºC for 10 minutes4ºC forever
?. [Amplification of Self-Reporting Transcripts]
At this point, gel electrophoresis can be performed to check the quality of amplification. We recommend running 5 uL of the PCR product from step 23 on 1% TAE agarose gel. The expected product is a smear extending from ~1 kb up to 5 kb.As a control, we recommend that the constitutive β-actin gene be amplified in parallel to the calling card libraries in steps 22 and 23. The control amplification uses the same PCR mix and thermocycler settings as step 22 and 23, but replaces the calling card forward and reverse primers with human β-actin primers (sequence provided in Materials as Raff_ACTB_F and Raff_ACTB_R). The expected product of the B-actin amplification is 626 bp (see Figure 1 in https://doi.org/10.2144/97233st02).
1% TAE gel showing expected products of SRT amplification. Left: the first 12 lanes are biological replicates of a calling card experiment, while the thirteenth is a no template control. The calling card libraries appear as smears extending up to 5 kb. Right: amplification of β-actin with Raff_ACTB_F and Raff_ACTB_R from the same RT product as the SRT samples produces the expected 626-bp product. The ladder is NEB's 1 kb Plus (previously, 2-Log) DNA Ladder (#N3200S).
?. [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 each 50 µl PCR mixture (0.6x ratio; if you did a half-size PCR, add 15 µl beads). 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 2 minutes. Aspirate supernatant and discard.
?. [Purification of PCR Products]
Add 200 µl of freshly-prepared 70% ethanol and incubate ≥ 30 seconds. Aspirate supernatant and discard.
?. [Purification of PCR Products]
Repeat Step #27.
?. [Purification of PCR Products]
Air dry the pellet at room temperature for 2 minutes.
?. [Purification of PCR Products]
Remove the tube from the magnetic rack. Add 20 µl ddH2O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes.
?. [Purification of PCR Products]
Place on magnetic rack for 1 minute, or until supernatant is clear.
?. [Purification of PCR Products]
Transfer supernatant to new tube. Create a 1:10 dilution and quantitate using the Qubit dsDNA HS Assay Kit.
Expected concentration of product should be 10-20 ng/µl.
?. [Generation of Bulk Calling Card Libraries]
The tagmentation protocol fragments the long PCR products into libraries suitable for sequencing. This protocol is based on the standard Drop-seq library preparation workflow. Continue processing each replicate independently.
?. [Generation of Bulk Calling Card Libraries]
Preheat thermocycler to
?. [Generation of Bulk Calling Card Libraries]
Take 1 ng of PCR product and resuspend in a total of 5 µl ddH2O in a PCR strip tube.
?. [Generation of Bulk Calling Card Libraries]
Add 10 µl of Nextera Tagment DNA (TD) Buffer and 5 µl of Amplicon Tagment Mix (ATM). Pipette to mix and briefly spin down; bubbles are normal. Incubate at for
?. [Generation of Bulk Calling Card Libraries]
Add 5 µl of Neutralization Tagment (NT) Buffer. Pipette to mix and briefly spin down; bubbles are normal. Incubate at room temperature for
?. [Generation of Bulk Calling Card Libraries]
Add the following to each PCR tube in order:15 µl Nextera PCR Mix (NPM)8 µl ddH2O1 µl of 10 µM barcoded piggyBac primer (e.g. OM-PB-ACG)1 µl of 10 µM indexed Nextera N7 primer (e.g. Nextera_N701)Each replicate should be identifiable by its barcode-index combination. It would be ideal if each replicate had a unique barcode and a unique index assigned to it. For some experimental setups, that may not be feasible. One option might be to assign a different index for different conditions/treatments, and within a condition/treatment, assign different barcodes to each replicate.
?. [Generation of Bulk Calling Card Libraries]
Perform PCR using the following thermocycling parameters:95ºC for 3 minutes13 cycles of:    95ºC for 10 seconds    50ºC for 30 seconds    72ºC for 30 seconds72ºC for 5 minutes4ºC forever
?. [Generation of Bulk Calling Card Libraries]
Purify PCR libraries using AMPure XP beads. Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.
?. [Final Quantitation and Sequencing]
Create a 1:10 dilution of each final library. Measure concentrations using the Qubit dsDNA HS Assay Kit or on a TapeStation device with a High Sensitivity D1000 ScreenTape. Libraries should be smoothly distributed between 300-60 bp.
Expected concentration of product should be 2-4 ng/µl.
?. [Final Quantitation and Sequencing]
Libraries can be sequenced on any Illumina sequencing platform. Due to the low complexity nature of calling card libraries, we recommed adding PhiX at a final concentration of 50%.
Bulk calling card libraries only use the information from read 1 for mapping insertions. Therefore, single-end sequencing should be sufficient, with at least 75 bp for read 1. An index 1 read will also be necessary for demultiplexing samples.
?. [Generation of Bulk Calling Card Libraries]
Add 35 µl beads to each 50 µl PCR mixture (0.7x ratio). Mix by pipetting 10 times until evenly dispersed.
?. [Generation of Bulk Calling Card Libraries]
Incubate at room temperature for
?. [Generation of Bulk Calling Card Libraries]
Place on a magnetic rack for 2 minutes. Aspirate supernatant and discard.
?. [Generation of Bulk Calling Card Libraries]
Add 200 µl of freshly-prepared 70% ethanol and incubate ≥ 30 seconds. Aspirate supernatant and discard.
?. [Generation of Bulk Calling Card Libraries]
Repeat Step #27.
?. [Generation of Bulk Calling Card Libraries]
Air dry the pellet at room temperature for 2 minutes.
?. [Generation of Bulk Calling Card Libraries]
Remove the tube from the magnetic rack. Add 11 µl ddH2O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes.
?. [Generation of Bulk Calling Card Libraries]
Place on magnetic rack for 1 minute, or until supernatant is clear. Transfer supernatant to new tube.
?. [cDNA Synthesis]
Create 1x Maxima RT buffer:For 5 or fewer samples, combine 1 uL of 5X Maxima RT buffer with 4 uL of ddH2O.Mix by pipetting and store on ice.
?. [cDNA Synthesis]
Create a 0.5x Maxima RT H Minus enzyme dilution:You will need 1 uL of the 0.5x enzyme dilution for every sample being processed. Avoid pipetting volumes