TITLE: 3&#39;-DGE High Throughput RNA Library Preparation
AUTHORS: Sarah Boswell, Caitlin Mills, Feodor Price, Stewart Rudnicki

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for the high-throughput transcriptome screening method called, 3’ Digital Gene Expression (3’-DGE). In this method RNA is purified from cell lysate and cDNA is generated by oligo dT priming, during which unique molecular identifiers (UMIs) are incorporated and strand-specificity is preserved. The 3’-DGE protocol has been optimized for 384 well plate experiments with a few thousand cells per well. The 3’-DGE method and a similar method known as DRUG-seq have been shown to be able to recapitulate 85% of the transcriptome you can obtain with standard mRNA-seq. In our experience using 3’-DGE we sequence each well at a low depth, aiming for ~1 million reads per sample which allows us to asses 40-80% of the transcriptome depending on the cell type. It is possible to sequence these samples to a greater depth if warranted. It is important to keep in mind that in 3’-DGE, and most low-input methods, only the 3’ poly-A end of the transcript is preserved. Therefore, this technique cannot be used if it is important to capture splice-isoforms or transcripts that do not have a poly-A tail.</div><div class = "text-block">This is a modification of the SCRB-seq protocol originally published here:</div><div class = "text-block"><a href="https://www.biorxiv.org/content/10.1101/003236v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/003236v1</span></a><span>; </span></div><div class = "text-block">Recognition also goes to the Enard group and their modifications to SCRB-seq found here:</div><div class = "text-block"><a href="https://www.protocols.io/view/mcscrb-seq-protocol-p9kdr4w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/mcscrb-seq-protocol-p9kdr4w</span></a></div><div class = "text-block">The home-brew SPRI beads used in this study are made with this protocol:</div><div class = "text-block"><span style = ":;"> </span><a href="https://dx.doi.org/10.17504/protocols.io.bkppkvmn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.bkppkvmn</span></a></div></div>

[STEPS]
?. [Cell Plating]
Cell plating conditions will vary depending on your experiment and cell type. Usually 2,000-10,000 cells per well.For best compatibility with ICCB-L robotics please use GREINER MICROPLATE, 384 WELL, PS, F-BOTTOM, µCLEAR now sold as Perkin Elmer CellCarrier ULTRA
This protocol has only been tested with adherent mammalian cells.Note that you should ONLY plate cells in wells you want data from. Any unused wells need to be empty of cells.  PBS or Media is fine, but no cells.If you are setting this up on your own robotic system you can use a different plate. Just be consistent with the plates used for proper robotic head height.
?. [Cell Lysis]
Purified RNA or intact adherent cells in 384 well format can be used for 3'-DGE.  (Note, we have had better experience with starting from intact cells.)1. If starting with purified RNA, skip to Step 22.2. If starting with intact cells, proceed to Step 3.3. Lysate from 4 x 96 well plates can also be used. 96 well plates are re-arrayed to 384 well format in the first stage of RNA purification.
?. [Cell Lysis]
Prepare enough Lysis Buffer for the number of samples.  (Protocol is for 384 well format)    TCL buffer + 1 % ?-mercaptoethanol  ABC1ReagentWellPlate2TCL Buffer9.9 µL4.22 mL3ß-mercaptoethanol0.1 µL42.2 µl4Total10 µL4.22 mL
ABC1ReagentWellPlate2TCL Buffer9.9 µL4.22 mL3ß-mercaptoethanol0.1 µL42.2 µl4Total10 µL4.22 mL
?. [Cell Lysis]
Aspirate media and wash wells once with PBS.We do this with a Biotek plate washer:
?. [Cell Lysis]
Remove most of the PBS. Our robotic system usually leaves behind about 10 µl of PBS.
?. [Cell Lysis]
Addof lysis buffer to each well of your 384-well plate.
10 µl
?. [Cell Lysis]
Incubate for  on belly dancer to aid in lysis.  Freeze  until ready to proceed with RNA extraction.
25 Room temperature
-80 °C
Incubation can be 0-30 min. Be consistent with your samples.
?. [Safe Stop Point]
Lysate plate can be stored at  until RNA extraction. Use within a few weeks of lysis is best, but we have stored samples for months with successful extraction.
-80 °C
?. [Set up for RNA purification on BRAVO]
Bring 384 well lysate plate to ICCB-L with the following reagents:Home Brew SPRI Beads  dx.doi.org/10.17504/protocols.io.bkppkvmn ()Fresh 80% Ethanol ()Water ()Reagent Reservoirs384 well multichannel pipette & TipsSet of standard pipettes & Filter tipsQubit        instrument        reagents & standards        tubesRepeater & 2x 1 mL tips1x Marsh waste plate3x Marsh plates for reagents2x Eppendorf Twintec PCR plates (need 2x more for RT step)3x BRAVO tip boxes for reaction (need 2.25x more for RT step)        instrument        reagents & standards        tubesRepeater & 2x 1 mL tips1x Marsh waste plate3x Marsh plates for reagents2x Eppendorf Twintec PCR plates (need 2x more for RT step)3x BRAVO tip boxes for reaction (need 2.25x more for RT step)
15 mL
50 mL
10 mL
?. [Set up for RNA purification on BRAVO]
Fill 3 separate clean MARSH plates with:    1)  SPRI beads/well    2)  80% Ethanol    3)  RNAse free waterWe use a 384 well multichannel (16 channel) pipette to make this easier.
40 µl
75 µl
35 µl
*Important - seal water plate and spin down   1000 xg.      Removes any bubbles that form under the water in the plate.If purifying multiple plates, can double the volume of water.  Other plates need to be fresh for each reaction.
?. [Set up for RNA purification on BRAVO]
Thaw lysate plate in the hood for around .Spin down lysate  plate and water plate  1000 xg before opening seal.
?. [Set up for RNA purification on BRAVO]
Set up reagents and plates on BRAVO as follows:
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Load tips #1 [Fluotics AGI-30.NS].Mix cell lysate (WATCH ROBOT – be sure can see efficient mixing).Transfer  of cell lysate to a 384-well Eppendorf Twintec PCR plate.             This is the "Reaction Plate".
10 µl
Need to watch during mixing. If lysate mixing is not achieved reaction will not work.  If a different plate was used for cell plating you may need to adjust the height of tips on the BRAVO.
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Mix SPRI beads in bead source plate ( 2x) Add  of beads to the reaction plate and mix ( 5x).         This is a 1.5x SPRI clean. Ratio of sample to beads is 1:1.5.Incubate  for .
25 µl
28 µl
15 µl
-80 Room temperature
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Incubate  for . (TAPE DOWN PLATE ON MAGNET)
-80 Room temperature
Need to tape plate onto magnet or it gets pulled up by Bravo head.
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Load tips #2 [Fluotics AGI-30.NS]. Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads with   of 80% EtOH (  delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads a second time with  of 80% EtOH ( delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads with   of 80% EtOH (  delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads a second time with  of 80% EtOH ( delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads with  of 80% EtOH ( delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads a second time with  of 80% EtOH ( delay before removing the liquid).Leaving the reaction plate on the magnet, remove liquid and dispense into the waste plate. Wash the beads a second time with of 80% EtOH (delay before removing the liquid).
25 µl
25 µl
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Perform one last aspirate to remove all of the EtOH from the reaction plate.Dry beads for .
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
While the reaction plate is still on the magnet, add  of nuclease free water.(Remove tape from magnet and manually separate plate from magnet if stuck.)
20 µl
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Remove the reaction plate from the magnet and resuspend the beads.( 15x at a height of 3 mm from the bottom of the plate; 3x). Look at plate after mixing and be sure it looks well mixed.
15 µl
If mixing is problematic you can take plate off the Bravo, seal it, smack it down then vortex it. Then quick spin followed by another mix step on the Bravo.If the SPRI beads look clumpy/sandy when mixing it is possible the extraction failed due to an issue with the lysis buffer, beads or ethanol. Heating the plate up to 50°C can help with the elution. This should be a last resort option.  If samples this hard to dissolve / look clumpy it may be worth aborting the experiment unless           samples are precious
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Incubate for .Transfer the reaction plate to the magnet.Incubate   at  .Transfer all of the supernatant to a fresh 384-well Twintec PCR plate.Incubate  at .        This is the "Final Plate".
-80 Room temperature
-80 Room temperature
?. [BRAVO DGE program for RNA purification (step 1 of DGE program)]
Pick random wells to check RNA concentration by Qubit.  2-5 ng/µl is the goal  Use 2µl of sample to check concentration   If starting from 96 well plates concentrations are often much higher 5-10 ng/µl
Can also take some sample to run RNA pico BioAnalyzer to check RNA quality. This is usually done in the pilot 3'-DGE run. On the actual sample plate BioA is not usually run.
?. [Safe Stop Point]
RNA can be frozen here at  before moving on to RT/Library prep.
-80 °C
BEST to proceed directly to next step in reaction.Seal and spin RNA plate before using for RT step.
?. [Set up for reverse transcription on BRAVO]
Bring reagents for RT:Repeater & 2x 1 mL tips2x Eppendorf Twintec PCR plates 2.25x BRAVO tip boxes for reaction Set of standard pipettes & Filter tips5x RT Buffer                              850 µldNTPs (NEB - #N0447L )        425 µlE5V6NEXT (10uM)                   425 µlMaxima H- RT Enzyme (Thermo Scientific #EP0753)     50 µl
?. [Set up for reverse transcription on BRAVO]
Make master mix  ABC1ReagentWellPlate2Maxima H Minus RT0.125 µl50 µl3Maxima RT Buffer (5x)2 µl850 µl4dNTPs (25 mM)1 µl425 µl5E5V6NEXT (10 uM)1 µl425 µl6Total4.125 µl1750 µlDispense  master mix into the first 3 columns of a 384-well eppendorf twintec plate.This is your "Master Mix Plate".
ABC1ReagentWellPlate2Maxima H Minus RT0.125 µl50 µl3Maxima RT Buffer (5x)2 µl850 µl4dNTPs (25 mM)1 µl425 µl5E5V6NEXT (10 uM)1 µl425 µl6Total4.125 µl1750 µl
36 µl
?. [Set up for reverse transcription on BRAVO]
Thaw the RNA plate and the E3V6Next adapter plate ()  in hood.Spin down RNA and adapter plates  1000 xg.
10 µl
?. [Set up for reverse transcription on BRAVO]
Set up reagents and plates on BRAVO as follows:
If using RNA as input, make sure to normalize concentrations (minimum: 0.5 ng/µl, optimum 20 ng/µl) across samples.
?. [BRAVO DGE program for RT (step 2 of DGE program)]
Using the BRAVO Add  of the master mix to each well of the "PCR plate".Change tips after every 4 dispense steps.
4.125 µl
There is JUST barely enough master mix for a whole plate.  In the last dispense watch to make sure enough master mix went into all wells.  It is sometimes necessary to dispense into a few wells by hand.
?. [BRAVO DGE program for RT (step 2 of DGE program)]
Add  of the unique barcoded E3V6NEXT adapters () per well from the Primer plate to the PCR Plate. (Be sure the adapter plate is taped down.)
1 µl
?. [BRAVO DGE program for RT (step 2 of DGE program)]
Using the BRAVO, transfer  (assume ~5 ng/µl) of the RNA from the RNA plate to the PCR Plate. (Be sure the RNA plate is taped down to the magnet.)Mix  3x
4.875 µl
5 µl
It is important the RNA plate is on the magnet for 1-2 minutes before transferring. There are often residual SPRI beads in the RNA plate that you do not want carried over into the PCR plate for the RT reaction.
?. [BRAVO DGE program for RT (step 2 of DGE program)]
Seal the plate and bring to  incubator.  This could be done in qPCR machine.  We use an air incubator with a metal block pre-equilibrated in the incubator for even heating.
42 °C
Block:  Labnet D12384 Aluminum Dry Bath Dual Block, Holds 384 Well PCR Plate
?. [BRAVO DGE program for RT (step 2 of DGE program)]
Incubate for  at .
42 °C
?. [cDNA Pooling & Purification]
Fit 384 well plate upside down onto collection plate (VBlok reservoir from Clickbio VBLOK200).
?. [cDNA Pooling & Purification]
Spin 1,000 xg for .
?. [cDNA Pooling & Purification]
Collect cDNA into 50 mL Falcon tube. Expected volume ~3.2-3.6 mL.
?. [cDNA Pooling & Purification]
Add 5x volume Qiagen PB buffer (3.4 mL = 17 mL, 3.6 mL = 18 mL)   Add  directly to a 50 mL Falcon tube.   Use the rest to rinse out collection plate then pipette into the 50 mL Falcon tube with the rest of the sample.
10 mL
?. [cDNA Pooling & Purification]
Load the sample onto QIAquick PCR purification column using vacuum filter system.     The loading will slow down as more sample is loaded.
Jam the luer-lock end of a 3 or 5 mL syringe barrel onto the column to make a "tube extender" to allow for easier loading of the column.
?. [cDNA Pooling & Purification]
Once all the sample has been loaded, wash the syringe barrel column with  of PE buffer.
750 µl
?. [cDNA Pooling & Purification]
Remove the syringe barrel column and wash the column once more with  of PE buffer.
750 µl
?. [cDNA Pooling & Purification]
Dry the column by centrifuging at >10,000 xg for .
?. [cDNA Pooling & Purification]
Elute cDNA with of nuclease free water (usually with water warmed to ~.    Let water sit on column for , then spin at >10,000 xg for .
21 µl
60 °C
?. [cDNA Pooling & Purification]
Check concentration of sample using dsDNA HS Qubit Assay.   Expected yield 30-80 ng/µl.
Do NOT pause protocol here.  Proceed through Exo I treatment and amplification before pausing protocol.
?. [Exonuclease I Treatment]
Mix  of purified cDNA with  of Exonuclease I Buffer (10x) and  of Exonuclease I. Incubate as follows:  ABC1StepTemperatureTime2Incubation37˚C30 min3Heat Inactivation80˚C20 min
17 µl
2 µl
1 µl
ABC1StepTemperatureTime2Incubation37˚C30 min3Heat Inactivation80˚C20 min
?. [Full length cDNA Amplification]
Prepare Amplification Mix  AB1Reagent1x2cDNA from previous step20 µl310x Advantage 2 PCR buffer5 µl4dNTPs1 µl5SINGV6 primer (10 µM)1 µl6Advantage 2 Polymerase Mix1 µl7H2022 µl8Total50 µl
AB1Reagent1x2cDNA from previous step20 µl310x Advantage 2 PCR buffer5 µl4dNTPs1 µl5SINGV6 primer (10 µM)1 µl6Advantage 2 Polymerase Mix1 µl7H2022 µl8Total50 µl
You can also use KAPA HiFi 2x ReadyMix.    If using KAPA mix, change the   steps below to  .
?. [Full length cDNA Amplification]
Incubate the Pre Amplification PCR as follows:  ABCD1StepTemperatureTimeCycles2Initial Denaturation95˚C1 min1 cycle3Denaturation95˚C15 sec5 cycles*4Annealing65˚C30 sec5Extension68˚C6 min6Final Extension72˚C10 min1 cycle7Storage4˚Cforever
ABCD1StepTemperatureTimeCycles2Initial Denaturation95˚C1 min1 cycle3Denaturation95˚C15 sec5 cycles*4Annealing65˚C30 sec5Extension68˚C6 min6Final Extension72˚C10 min1 cycle7Storage4˚Cforever
*For a 384 well plate 5 cycles is enough.For 96 well plate, run 10 cycles.If using KAPA HiFI mix, change   steps to  .
?. [Safe Stop Point]
Can leave reaction at  overnight.
4 °C
?. [cDNA Bead Purification (0.7x)]
Dilute PCR mix with  Water for 100 µl total reaction volume.
50 µl
?. [cDNA Bead Purification (0.7x)]
Mix sample with  Ampure XP beads at a ratio of 1:0.7        Be sure Ampure  beads are at Room Temperature and fully resuspended before use.
70 µl
Ampure XP is used instead of homebrew SPRI beads in order to ensure best size selection at this critical stage where we are removing any primer dimers.
?. [cDNA Bead Purification (0.7x)]
Incubate for  at .
20 Room temperature
?. [cDNA Bead Purification (0.7x)]
Place the tube on the magnet stand until clear and discard supernatant.
?. [cDNA Bead Purification (0.7x)]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant.
500 µl
?. [cDNA Bead Purification (0.7x)]
Repeat wash step once more.      Use aspirator to remove residual ethanol to speed up drying time.
?. [cDNA Bead Purification (0.7x)]
Air dry beads for .
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads to avoid over-drying.
?. [cDNA Bead Purification (0.7x)]
Take sample off the magnet and elute cDNA in H2O.
16 µl
?. [Safe Stop Point]
Can leave sample at  until ready to continue.
-20 °C
?. [cDNA Quantification and Quality Check]
Quantify the cDNA using the Quant-iT PicoGreen dsDNA assay kit or dsDNA HS Qubit following the manufacturer's protocol.  Note will QC again with Qubit after diluting sample for Nextera XT protocol.    Use  of clean cDNA for quantification.             Usually ~15-60 ng/µl
1 µl
?. [cDNA Quantification and Quality Check]
Optional: Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.(Usually do not bother with this step, but can be informative when you are new to the protocol.)
?. [Tagmentation (Nextera XT)]
Prepare 20ul of a 1 ng/µL solution of cDNA for Nextera XT Tagmentation.     Use 5ul to check concentration by Qubit.Further dilute sample to 0.8ng/ul for Nextera XT protocol.
Can also use Nextera FLEX kit instead of XT kit.  Use mid range amount of sample input ~250ng and  use 1/2 volume reactions. Follow manual instructions performing x5 cycles of amplification. For the final purification, follow the protocol which includes a double sided SPRI selection (0.5x followed by additional 1x).
?. [Tagmentation (Nextera XT)]
Prepare Tagmentation Mix (enough for 5 replicate reactions).NOTE - add Amplicon Tagment Mix last (ATM)  ABC1Reagent1x5x2cDNA (0.8 ng/µL)1 µL5 µl3Tagment DNA Buffer10 µL50 µl4H2O4 µL20 µl5Amplicon Tagment Mix5 µL25 µl6Total20 µL100 µl
ABC1Reagent1x5x2cDNA (0.8 ng/µL)1 µL5 µl3Tagment DNA Buffer10 µL50 µl4H2O4 µL20 µl5Amplicon Tagment Mix5 µL25 µl6Total20 µL100 µl
Do tagmentation reaction in replicates of 5 in a PCR strip.
?. [Tagmentation (Nextera XT)]
Distribute  of Tagmentation Mix in 5 reactions in PCR strip and incubate the reaction for  at .
20 µl
55 °C
?. [Tagmentation (Nextera XT)]
Add  NT Buffer to each reaction in PCR strip, mix, and incubate for  at .
5 µl
20 Room temperature
?. [Nextera Index PCR]
Nextera Index PCR Mix  ABC1Reagent1x5x2Indexing primer N7XX (5 µM)0.5 µL2.5 µl3P5NextPT5 (5 µM)0.5 µL2.5 µl4NPM PCR Mix15 µL75 µl5H2O9 µL45 µl6Total25 µL125 µl
ABC1Reagent1x5x2Indexing primer N7XX (5 µM)0.5 µL2.5 µl3P5NextPT5 (5 µM)0.5 µL2.5 µl4NPM PCR Mix15 µL75 µl5H2O9 µL45 µl6Total25 µL125 µl
?. [Nextera Index PCR]
Add  Nextera Index PCR mix to each replicate and incubate as follows:  ABCD1StepTemperatureTimeCycles2Gap Fill 72˚C3 min1 cycle3Initial Denaturation95˚C30 sec1 cycle4Denaturation95˚C10 sec12 cycles5Annealing55˚C30 sec6Elongation72˚C1 min7Final Elongation72˚C1 min1 cycle8Hold4˚C∞
25 µl
ABCD1StepTemperatureTimeCycles2Gap Fill 72˚C3 min1 cycle3Initial Denaturation95˚C30 sec1 cycle4Denaturation95˚C10 sec12 cycles5Annealing55˚C30 sec6Elongation72˚C1 min7Final Elongation72˚C1 min1 cycle8Hold4˚C∞
?. [Library Bead Purification]
Pool all 5 PCR Replicates; rinse PCR wells with  of water (total). Combine with pooled sample for a total of  of sample.
50 µl
300 µl
?. [Library Bead Purification]
Mix Index PCR with  Ampure XP beads at a a ratio of 1:0.8
240 µl
If doing a gel cleanup use a 1:1.2 ratio to capture as much cDNA since the primers will be cut out from the gel.
?. [Library Bead Purification]
Incubate for  at .
20 Room temperature
?. [Library Bead Purification]
Place the tube on the magnet stand until clear and discard supernatant.
?. [Library Bead Purification]
Wash with  of 80% EtOH while the tube is on the magnet. Discard the supernatant.
500 µl
?. [Library Bead Purification]
Repeat wash step once more.
?. [Library Bead Purification]
Air dry beads for  in  air incubator then finish drying at RT.  You can dry at RT only, but will take much longer to dry.
37 °C
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
?. [Library Bead Purification]
Remove tube from the magnet and elute library in  H2O.
15 µl
?. [Final Sample QC]
Quantify sample using Qubit dsDNA HS.  Expected yield is low 1-4 ng/µl (which is ~4 nM).If yields are much higher could cycle for fewer cycles during final amplification.
?. [Final Sample QC]
Run sample on Tapestation or Bioanalyzer to assess size.  Below is an example from the tapestation.
?. [Safe Stop Point]
Store sample at  until sequenced.
-20 °C
?. [Sequencing]
We have found that running a single 384 well DGE plate at a time is best for clustering on a NextSeq.  For a NovaSeq run, multiple plates can be pooled together.These libraries are difficult to cluster appropriately: usually you need to load almost 2x the concentration you would normally run to get good clustering. (Test this in your own hands!)
?. [Sequencing]
Libraries are run in a paired-end manner:17 cycles Read 1                            Well index and UMI8 cycles Index 1                             Nextera Plate Index60 (or more) cycles Read 2          cDNA Fragment
?. [Sequencing]
BCL file to FASTQ file conversion is a bit tricky due to the short length of Read 1. Speak to your sequencing core to ensure you get your data correctly.  The code below has worked for our team:bcl2fastq --adapter-stringency 0.9 --barcode-mismatches 0 --fastq-compression-level 4 --ignore-missing-bcls --ignore-missing-filter --ignore-missing-positions --min-log-level INFO --minimum-trimmed-read-length 0 --sample-sheet /n/boslfs/INSTRUMENTS/illumina/190107_NB501677_0376_AHTLM5BGX7/SampleSheet.csv --runfolder-dir /n/boslfs/INSTRUMENTS/illumina/190107_NB501677_0376_AHTLM5BGX7 --output-dir /n/boslfs/ANALYSIS/190107_NB501677_0376_AHTLM5BGX7 --processing-threads 8 --no-lane-splitting --mask-short-adapter-reads 0 --use-bases-mask y17,i8,y46
?. [BRAVO DGE program for RT (step 2 of DGE program)]
After incubation give plate a quick spin 1000xg Then carefully remove the seal
?. [cDNA Pooling & Purification]
After Inverting the Falcon tube to mix, give it a quick spin 1000xg
?. [Library Bead Purification]
Incubate beads  at RT
?. [Library Bead Purification]
Put tube with eluted beads back on magnet for 2min Remove  for a final libraryUse remining volume on beads for QC steps below
12 µl
?. [cDNA Bead Purification (0.7x)]
Incubate sample for  at RT to elute the cDNA
?. [cDNA Bead Purification (0.7x)]
Then place the sample back on the magnet and remove  .  Use residual sample on beads to quantitate sample with Qubit.
14 µl