TITLE: Probe-based target enrichment of SARS-CoV-2
AUTHORS: Mariateresa De Cesare

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Viral RNA library prep using </span><span style = "font-weight:bold;">SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian </span><span>followed by a probe-based bait capture (SeqCap, Roche and xGen, IDT) to generate tagged enriched viral libraries from total RNA that retain directionality in the library. </span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>The </span><span style = "font-weight:bold;">SMARTer Stranded </span><span>kit uses random primers, tailed with Illumina Read1 sequence, to start reverse transcription, and a Template Switching Oligo (TSO) to add Read2 sequence at the 3’ of the synthesized cDNA. The 1</span><span style = "vertical-align:super;">st</span><span> stranded cDNA is then amplified using Indexed primers to generate in one step 2</span><span style = "vertical-align:super;">nd</span><span> strand cDNA and complete tagged Illumina libraries. </span></li><li style = "counter-reset:ol0;list-style-type:disc;">Pooled libraries undergo target enrichment using custom virus-specific biotinylated probes to capture cDNA derived from viral RNA present in the sample.</li><li style = "counter-reset:ol0;list-style-type:disc;">This method has been optimized for large scale viral sequencing projects, and up to 2 x 96-well RNA plates can be prepped in parallel.</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>The protocol follows “</span><span style = "font-weight:bold;">Option 2 (without fragmentation)</span><span style = "font-weight:bold;font-weight:bold;">”</span><span style = "font-weight:bold;"> </span><span>workflow of the kit User Manual for library preparation, with ¼ of the recommended reaction volume for denaturation and cDNA synthesis, and ½ for PCR, all steps done in 384-well plates.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;font-style:italic;">NOTE:</span><span style = "font-style:italic;"> the kit contains a Ribodepletion PCR-based module not used in this protocol, so PCR reagents are in excess and a higher volume for PCR can be used safely without depleting the kit of reagents unevenly.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>The absence of an RNA fragmentation step coupled with more stringent cleanups (at 0.68x) generates libraries with longer insert size, therefor this protocol is only to be used on RNA samples of high quality, and unnecessary freeze-thaw of the RNA </span><span style = "font-weight:bold;">MUST </span><span>be avoided. As the RNA extracted from plasma/swabs is below the detection level for QC, if in doubt about the quality of the RNA provided, proceed with library preparation and pooling, but check the size of the libraries before cleanup of the pool as the 0.68X beads ratio recommended in this protocol would remove most of the library if the starting RNA is of lower quality.</span></li><li style = "counter-reset:ol0;list-style-type:disc;">To maximize RNA input and improve sensitivity of the assay, an RNA concentration step at the start of the procedure has been added, though not necessary to generate libraries.</li></ul></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;font-style:italic;">NOTE:</span><span style = "font-style:italic;"> the kit contains a Ribodepletion PCR-based module not used in this protocol, so PCR reagents are in excess and a higher volume for PCR can be used safely without depleting the kit of reagents unevenly.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>The absence of an RNA fragmentation step coupled with more stringent cleanups (at 0.68x) generates libraries with longer insert size, therefor this protocol is only to be used on RNA samples of high quality, and unnecessary freeze-thaw of the RNA </span><span style = "font-weight:bold;">MUST </span><span>be avoided. As the RNA extracted from plasma/swabs is below the detection level for QC, if in doubt about the quality of the RNA provided, proceed with library preparation and pooling, but check the size of the libraries before cleanup of the pool as the 0.68X beads ratio recommended in this protocol would remove most of the library if the starting RNA is of lower quality.</span></li><li style = "counter-reset:ol0;list-style-type:disc;">To maximize RNA input and improve sensitivity of the assay, an RNA concentration step at the start of the procedure has been added, though not necessary to generate libraries.</li></ul></div></div>

[STEPS]
?. [Preparations]
Bring the RNAClean XP (A63987) to .
0 Room temperature
?. [Preparations]
Ensure a chilling block to accommodate 384-well plates is at .
-20 °C
?. [Preparations]
Defrost reagents (SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian and SMART TSO Mix v2) .
on ice
?. [Preparations]
Prepare the following program on a 384 thermal cycler:  AB1TemperatureTime272 °C∞372 °C3 min442 °C∞542 °C90 min670 °C10 min74 °C∞
AB1TemperatureTime272 °C∞372 °C3 min442 °C∞542 °C90 min670 °C10 min74 °C∞
?. [Concentration of the RNA using magnetic beads]
Thaw the RNA samples  (up to 2 plates of 96 samples).
on ice
This protocol describes how process 2 plates of 96 samples in parallel, and uses a Gilson PlateMaster. If a PlateMaster or equivalent is not available, it is not recommended to process more than one 96-well plate.For 96 or fewer samples, the protocol can be executed with multichannel pipettes.
?. [Concentration of the RNA using magnetic beads]
Add in RNAClean XP beads at 1.8 ratio to all RNA samples.
NOTE: Precise measurement of the RNA volume of each sample is not crucial at this stage, as this “cleanup” has the only purpose of concentrating the RNA, and not removing unwanted oligos
?. [Concentration of the RNA using magnetic beads]
Incubate at  for .
-20 Room temperature
?. [Concentration of the RNA using magnetic beads]
Place the plate on magnet: allow beads to separate for .
?. [Concentration of the RNA using magnetic beads]
Remove supernatant and put aside in a new 96-well plate.
?. [Concentration of the RNA using magnetic beads]
Add  to wash the beads, incubate for .
[80% ethanol]
?. [Concentration of the RNA using magnetic beads]
Remove 80% EtOH.
?. [Concentration of the RNA using magnetic beads]
Repeat the ethanol wash (steps 10 – 11)
If two 96-well plates are being prepared together, do both ethanol washes on the 1st plate before moving to the 2nd plate, so that the same PlateMaster tips can be used for both washes on a plate.WARNING: Keep to time with washes on the 2nd plate to avoid over drying beads on the 1st!
?. [Concentration of the RNA using magnetic beads]
Briefly spin the plate and remove additional ethanol with fresh tips. Ensure any visible quantities of ethanol are removed.
?. [Concentration of the RNA using magnetic beads]
Remove the plate(s) from magnet and resuspend beads in .
[EB]
?. [Concentration of the RNA using magnetic beads]
Leave the beads in EB for  at .
-20 Room temperature
?. [Concentration of the RNA using magnetic beads]
During the 5 mins incubation, prepare a 384-well plate containing .
[SMART Pico Oligos Mix v2]
In our lab we use a Labcyte Echo 525 to dispense the Oligo Mix v2.If accuracy of pipetting such small volume is a concern, consider scaling up the volume of the all protocol from 1/4 to 1/2 or more.
?. [RNA denaturation]
Pre-heat the thermocycler to .
72 °C
?. [Concentration of the RNA using magnetic beads]
Add  to wash the beads, incubate for .
[80% ethanol]
?. [Concentration of the RNA using magnetic beads]
Remove 80% EtOH.
?. [RNA denaturation]
Once the 5 mins elution time is passed, place the RNA plate on the magnet and allow beads to separate for .
?. [RNA denaturation]
Transfer  into the plate containing .
[sample]
[freshly-dispensed SMART Pico Oligos Mix v2]
?. [RNA denaturation]
Mix with PlateMaster, quickly spin down the plate.
?. [RNA denaturation]
Final plate for denaturation should contain:  AB1ReagentVolume (μl)2SMART Pico Oligos Mix v20.253RNA24Total2.25 μl / reaction
AB1ReagentVolume (μl)2SMART Pico Oligos Mix v20.253RNA24Total2.25 μl / reaction
?. [RNA denaturation]
Place plate containing RNA and SMART Pico Oligos Mix v2 onto the pre-set thermocycler and incubate at  for .
72 °C
?. [RNA denaturation]
Immediately move samples to the chilling block and leave for .
?. [RNA denaturation]
Proceed immediately to First-strand cDNA synthesis
?. [First-strand cDNA synthesis]
Pre-heat thermal cycler to .
42 °C
?. [First-strand cDNA synthesis]
Prepare First Strand reverse transcription mastermix by mixing the following reagents in the order shown (no. reactions + 10%):  AB1ReagentVolume per reaction (μl)25X First-Strand Buffer13SMART TSO Mix v21.1254RNase Inhibitor0.1255SMARTScribe Reverse Transcriptase0.56Total2.75 μl
AB1ReagentVolume per reaction (μl)25X First-Strand Buffer13SMART TSO Mix v21.1254RNase Inhibitor0.1255SMARTScribe Reverse Transcriptase0.56Total2.75 μl
?. [First-strand cDNA synthesis]
Add  of MasterMix to each sample, mix by pipetting and then spin down the plate.
2.75 µl
?. [First-strand cDNA synthesis]
Place in the pre-heated thermocycler for first strand RT incubation:  for  →  for  →  hold.
42 °C
70 °C
4 °C
?. [First-strand cDNA synthesis]
Leave samples at  until next step.
4 °C
?. [First-strand cDNA synthesis]
SAFE STOPPING POINT!  at  or store at .
4 °C
-20 °C
?. [PCR amplification of cDNA and library generation]
Prepare thermocycler in a post-PCR area for second strand amplification.
?. [PCR amplification of cDNA and library generation]
Set up the PCR as follow:  ABC1ComponentVolume per reaction (μl)2SampleFirst-strand cDNA53EACHIndexed i7 primer (6.25 μM)14Indexed i5 primer (6.25μM)15Master Mix (MM)Nuclease-free Water56SeqAmp CB PCR Buffer (2X)12.57SeqAmp DNA Polymerase0.58Total5 samples + 2 primers + 18 MM = 25 μl / reaction
ABC1ComponentVolume per reaction (μl)2SampleFirst-strand cDNA53EACHIndexed i7 primer (6.25 μM)14Indexed i5 primer (6.25μM)15Master Mix (MM)Nuclease-free Water56SeqAmp CB PCR Buffer (2X)12.57SeqAmp DNA Polymerase0.58Total5 samples + 2 primers + 18 MM = 25 μl / reaction
?. [PCR amplification of cDNA and library generation]
Mix by pipetting, spin down the plate and start PCR:  ABCD1StepTemperatureTimeNo. cycles2Initial Denaturation94 °C60 seconds13Denaturation98 °C15 seconds124Annealing55 °C15 seconds5Extension68 °C30 seconds6Final Extension68 °C2 minutes17Hold10 °C∞
ABCD1StepTemperatureTimeNo. cycles2Initial Denaturation94 °C60 seconds13Denaturation98 °C15 seconds124Annealing55 °C15 seconds5Extension68 °C30 seconds6Final Extension68 °C2 minutes17Hold10 °C∞
?. [PCR amplification of cDNA and library generation]
SAFE STOPPING POINT!  at  or store at .
4 °C
-20 °C
?. [Pooling, cleanup and QC]
Pool in a 1.5 ml LoBind tube equal volume of each sample to be captured together
NOTE: Library prepared with this protocol would yield on average 2-5 ng/μl: pool enough of each library for a final pool with >> 500 ng (3 μl per sample for a 96-plex is usually plenty).
?. [Pooling, cleanup and QC]
Accurately measured with a pipette the volume of the pool: this is crucial for correct size-selection of the libraries with Ampure XP beads.
?. [Pooling, cleanup and QC]
Add 0.68x Ampure XP to each pool and perform a cleanup
WARNING: The 0.68x ratio is to be used only on libraries from high quality RNA! If that is not the case, use 0.8x of Ampure XP instead.
?. [Pooling, cleanup and QC]
Mix thoroughly either by pipetting or vortexing.
?. [Pooling, cleanup and QC]
Incubate for  at .
-20 Room temperature
?. [Pooling, cleanup and QC]
Transfer tubes to magnet and incubate for .
?. [Pooling, cleanup and QC]
Remove and keep supernatant in a clean tube. Do not transfer any beads in the supernatant. If this is a risk, leave  behind with the beads.
?. [Pooling, cleanup and QC]
Wash beads with . Leave for .
[ 80% ethanol]
?. [Pooling, cleanup and QC]
Remove and discard the supernatant.
?. [Pooling, cleanup and QC]
Centrifuging tube briefly to collect any residual ethanol at the bottom of the well and pipette this off with a P10 or P20 pipette.
?. [Pooling, cleanup and QC]
Ensure removal of all ethanol.
?. [Pooling, cleanup and QC]
Air dry beads until the well looks dry and the beads are starting to crack.
?. [Pooling, cleanup and QC]
Resuspend beads in a volume of EB equal to the starting pool volume or less.
NOTE: Eluting in less EB will speed the drying of the pool in the SpeedVac
?. [Pooling, cleanup and QC]
Mix by pipetting or vortexing. If vortexing, leave for  before centrifuging.
?. [Pooling, cleanup and QC]
Return tubes to magnet for .
?. [Pooling, cleanup and QC]
Transfer the clean eluant to a fresh 1.5 ml LoBind tube.
?. [Pooling, cleanup and QC]
Quantify the libraries pool using Qubit® dsDNA HS Assay Kit (Cat#: Q32854).
?. [Pooling, cleanup and QC]
Check the size of the libraries pool on an Agilent Tapestation with a High Sensitivity D1000 Screen Tapes assay (Cat#: 5067-5584).
?. [Pooling, cleanup and QC]
Bring forward 500 ng from each pool for capture (next section) and store the remaining uncaptured pool.
?. [Pooling, cleanup and QC]
Repeat wash with ethanol.
?. [Pooling, cleanup and QC]
Wash beads with . Leave for .
[ 80% ethanol]
?. [Pooling, cleanup and QC]
Remove and discard the supernatant.
?. [Hybridize xGen Lockdown Probes to Target]
Turn on a heat block that takes 1.5 ml tubes, and let it equilibrate to .
95 °C
?. [Hybridize xGen Lockdown Probes to Target]
Defrost xGen® Universal Blockers (IDT), COT Human DNA (vial 1, Roche, SeqCap EZ Accessory Kit, stored at -20°C).
?. [Hybridize xGen Lockdown Probes to Target]
Bring the 2X Hybridization buffer (vial 5) and Hybridization Component A (vial 6) to  (Roche, SeqCap EZ Hybridization and Wash Kit, stored at -20°C).
0 Room temperature
?. [Hybridize xGen Lockdown Probes to Target]
Remove the xGen Lockdown Probes (IDT) from -20°C freezer and defrost .
on ice
The xGen Lockdown Probes are custom biotinylated oligos designed to capture cDNA libraries derived from the viral RNA.This protocol is based on our work with HIV, and has been validated for SARS-CoV-2 using a panel of probes designed by our lab.
?. [Hybridize xGen Lockdown Probes to Target]
In a 1.5 ml tube, add:  AB1ComponentAmount2Multiplex DNA Sample Library Pool500 ng3COT Human DNA5 μl4xGen® Universal Blockers - TS Mix2 μl
AB1ComponentAmount2Multiplex DNA Sample Library Pool500 ng3COT Human DNA5 μl4xGen® Universal Blockers - TS Mix2 μl
?. [Hybridize xGen Lockdown Probes to Target]
Dry down the contents of the tube (libraries + COT Human DNA + Blocking Oligos) using a SpeedVac with high temperature.
?. [Hybridize xGen Lockdown Probes to Target]
Once the pool is completely dry, resuspend in:  AB1ComponentAmount22X Hybridization buffer (vial 5)7.5 μl3Hybridization Component A (vial 6)3 μl
AB1ComponentAmount22X Hybridization buffer (vial 5)7.5 μl3Hybridization Component A (vial 6)3 μl
?. [Hybridize xGen Lockdown Probes to Target]
Vortex for  then spin down
?. [Hybridize xGen Lockdown Probes to Target]
Place the tube in the  heat block for  to denature the DNA.
95 °C
?. [Hybridize xGen Lockdown Probes to Target]
Spin down and transfer the content of the tube to a 0.2 ml PCR tube.
?. [Hybridize xGen Lockdown Probes to Target]
Add  and top up with water to a final volume of 15 μl.
[xGen Lockdown Probes]
?. [Hybridize xGen Lockdown Probes to Target]
Mix by pipetting.
?. [Hybridize xGen Lockdown Probes to Target]
The tube should contain the following:  AB1ComponentAmount2Multiplex DNA Sample Library Pool500 ng*3Cot-1 DNA5 μg*4xGen® Universal Blockers - TS Mix2 μl*52X Hybridization Buffer (vial 5)7.5μl6Hybridization Component A (vial 6)3 μl7xGen Lockdown Probes4 μl8Nuclease-Free Water0.5 μl9Total15 μl
AB1ComponentAmount2Multiplex DNA Sample Library Pool500 ng*3Cot-1 DNA5 μg*4xGen® Universal Blockers - TS Mix2 μl*52X Hybridization Buffer (vial 5)7.5μl6Hybridization Component A (vial 6)3 μl7xGen Lockdown Probes4 μl8Nuclease-Free Water0.5 μl9Total15 μl
?. [Hybridize xGen Lockdown Probes to Target]
Incubate hybridization reaction at  in a thermocycler (lid heated at ) for  (or ).
47 °C
57 °C
?. [Hybridize xGen Lockdown Probes to Target]
If proceeding after a 4 h hybridization, change the heat block’s temperature used in step 63 to .
47 °C
?. [Hybridize xGen Lockdown Probes to Target]
Allow time to equilibrate to the set temperature.
?. [Prepare wash buffers]
If proceeding after an overnight hybridization, turn on a heat block that takes 1.5 ml tubes to , and let it equilibrate to the set temperature.
47 °C
?. [Prepare wash buffers]
2 h before the end of the hybridization, dilute 10X Wash Buffers (I, II, III and Stringent) and 2.5X Bead Wash Buffer (Roche, SeqCap EZ Hybridization and Wash Kit, stored at -20°C) to create 1X working solutions.  ABCD1Buffer (μl)Water (μl)Final volume (μl)210X Wash Buffer I (vial 1)33297330310X Wash Buffer II (vial 2)22198220410X Wash Buffer III (vial 3)22198220510X Stringent Wash Buffer (vial 4)4439644062.5X Bead Wash Buffer (vial 7)220330550
ABCD1Buffer (μl)Water (μl)Final volume (μl)210X Wash Buffer I (vial 1)33297330310X Wash Buffer II (vial 2)22198220410X Wash Buffer III (vial 3)22198220510X Stringent Wash Buffer (vial 4)4439644062.5X Bead Wash Buffer (vial 7)220330550
?. [Prepare wash buffers]
For each capture reaction, preheat the following wash buffers to  in the heat block:
47 °C
[1X Wash Buffer I]
?. [Prepare wash buffers]
Equilibrate buffers at  for at least  before starting wash steps of the captured DNA (section "Wash streptavidin beads to remove unbound DNA" below).
47 °C
?. [Prepare the Streptavidin Dynabeads]
Allow Dynabeads M-270 Streptavidin (stored at 4°C) to equilibrate to  for  before use (~30minutes before the end of the hybridization).
4 Room temperature
?. [Prepare the Streptavidin Dynabeads]
Mix the beads thoroughly by vortexing for .
?. [Prepare the Streptavidin Dynabeads]
Aliquot  per capture into a single 1.5 ml tube (i.e., for 1 capture use 100 μl beads, for 2 captures use 200 μl beads, etc.).
[streptavidin beads]
?. [Prepare the Streptavidin Dynabeads]
Place the tube in a magnetic separation rack. Allow the beads to separate from the supernatant. Carefully remove and discard the clear supernatant ensuring that all of the beads remain in the tube.
?. [Prepare the Streptavidin Dynabeads]
Add  per 100 μl beads. Vortex for .
[1X Bead Wash Buffer]
?. [Prepare the Streptavidin Dynabeads]
Place the tube back in the magnetic rack to bind the beads. Allow the beads to separate from the supernatant. Carefully remove and discard the clear supernatant ensuring that all of the beads remain in the tube.
?. [Prepare the Streptavidin Dynabeads]
Repeat wash with 1X Bead Wash Buffer.
?. [Prepare the Streptavidin Dynabeads]
After removing the buffer following the second wash, add 1X the original volume of beads of 1X Bead Wash Buffer (i.e., for 100 μl beads, use 100 μl buffer) and resuspend by vortexing.
?. [Prepare the Streptavidin Dynabeads]
Transfer  of the resuspended beads into a new 0.2 ml tube for each capture reaction.
100 µl
?. [Prepare the Streptavidin Dynabeads]
Place the tube in a magnetic rack to bind the beads. Allow the beads to separate from the supernatant. Carefully remove and discard the clear supernatant ensuring that all of the beads remain in the tube.
?. [Prepare the Streptavidin Dynabeads]
Add  per 100 μl beads. Vortex for .
[1X Bead Wash Buffer]
?. [Prepare the Streptavidin Dynabeads]
Place the tube back in the magnetic rack to bind the beads. Allow the beads to separate from the supernatant. Carefully remove and discard the clear supernatant ensuring that all of the beads remain in the tube.
?. [Bind hybridized target to the streptavidin beads]
Take the samples pool from  out of the thermocycler (do not stop the program!) quickly spin down and transfer to the tube containing prepared streptavidin beads.
?. [Bind hybridized target to the streptavidin beads]
Mix thoroughly by pipetting up and down 10 times.
?. [Bind hybridized target to the streptavidin beads]
Place the tube back into the thermal cycler set to  and incubate for  (set heated lid at ) to bind the DNA to the beads.
47 °C
57 °C
?. [Bind hybridized target to the streptavidin beads]
Vortex the tube for  every 15 min to ensure that the beads remain in suspension.
?. [Wash streptavidin beads to remove unbound DNA]
Take the samples out of the thermal cycler and add  to the tube and vortex for  to mix.
[pre-heated 1X Wash Buffer I]
?. [Wash streptavidin beads to remove unbound DNA]
Spin down and transfer the mixture to a fresh low-bind 1.5 ml tube.
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant containing unbound DNA and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Add  and pipette up and down 10 times to mix.Incubate on the heat block at  for .
[preheated 1X Stringent Wash Buffer]
47 °C
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant containing unbound DNA and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Repeat wash with preheated 1X Stringent Wash Buffer.
?. [Wash streptavidin beads to remove unbound DNA]
Add  and vortex for  to mix.
[room temperature 1X Wash Buffer I]
?. [Wash streptavidin beads to remove unbound DNA]
Add  and pipette up and down 10 times to mix.Incubate at  for .
[preheated 1X Stringent Wash Buffer]
47 °C
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant containing unbound DNA and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Add  and vortex for  to mix.
[room temperature 1X Wash Buffer II]
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Add  and vortex for  to mix.
[room temperature 1X Wash Buffer III]
?. [Wash streptavidin beads to remove unbound DNA]
Place the tube in the magnetic separation rack. Allow the beads to separate from the supernatant. Using a pipette, remove the supernatant and discard.
?. [Wash streptavidin beads to remove unbound DNA]
Remove the tube from the magnetic rack and add  to resuspend the beads. Mix thoroughly by pipetting up and down 10 times.
[Nuclease-Free Water]
Do not pellet or remove the beads: The post-capture PCR is done on the beads
?. [Wash streptavidin beads to remove unbound DNA]
SAFE STOPPING POINT!  at  or store at .
4 °C
-20 °C
?. [Post-capture PCR]
Thaw the KAPA HiFi HotStart ReadyMix and the Post-LM-PCR Oligos from the SeqCap EZ Accessory Kits v2 (stored at -20°C).
?. [Post-capture PCR]
Set up the PCR as follow:  ABC1ComponentVolume2EACHCaptured library on beads253MASTER MIXKAPA HiFi HotStart ReadyMix (Roche)504Post-LM-PCR Oligos 1&2, 5μM (Roche)55Nuclease free water206Total75 +25 library =100 μl / reaction
ABC1ComponentVolume2EACHCaptured library on beads253MASTER MIXKAPA HiFi HotStart ReadyMix (Roche)504Post-LM-PCR Oligos 1&2, 5μM (Roche)55Nuclease free water206Total75 +25 library =100 μl / reaction
?. [Post-capture PCR]
PCR incubation:  ABCD1StepTemperatureTimeNo. cycles2Initial Denaturation98 °C45 seconds13Denaturation98 °C15 seconds124Annealing60 °C30 seconds5Extension72 °C30 seconds6Final Extension72 °C1 minute17Hold10 °C∞
ABCD1StepTemperatureTimeNo. cycles2Initial Denaturation98 °C45 seconds13Denaturation98 °C15 seconds124Annealing60 °C30 seconds5Extension72 °C30 seconds6Final Extension72 °C1 minute17Hold10 °C∞
?. [Post-capture PCR]
SAFE STOPPING POINT! Store at  or .
-20 °C
4 °C
?. [Post-capture PCR]
Perform Ampure XP clean-up with 0.68x ratio and elute into .
20 µl
WARNING: The 0.68x ratio is to be used only on libraries from high quality RNA! If that is not the case, use 0.8x of Ampure XP instead
?. [Post-capture PCR]
SAFE STOPPING POINT! Store at  or .
-20 °C
4 °C
?. [Captured Pool QC and 10nM-ing]
Quality check the captured pool using Qubit and Tapestation.
Pools concentrations is highly variable and dependent on samples viral load (
?. [Captured Pool QC and 10nM-ing]
Tapestation profile should have a long tail, but it’s dependent of the quality of the starting RNA. For high quality RNA, after two 0.68x Ampure XP cleanups, one pre- and one post-capture, the profile should look like this:
?. [RNA denaturation]
Take the chilling block out of the freezer during the incubation