TITLE: RNA-seq Library Construction with the KAPA mRNA HyperPrep Kit
AUTHORS: Amanda Scholes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A protocol for making multiplexed RNA-seq libraries using KAPA Biosystems mRNA HyperPrep Kit, optimized for </span><span style = "font-style:italic;">Saccharomyces cerevisiae</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [Prepping mRNA Capture Beads]
1. Resuspend the mRNA Capture Beads (stored at 4°C) by gently pipetting (try to prevent foaming).2. For each library, transfer 26.25 µL of beads (25 µL x 1.05) to an RNase-free 1.7 mL microcentrifuge tube (up to 24 total reactions per one tube).3. Place the tube with beads on a Surebeads Magnetic Rack (Bio-Rad #1614916) and wait for the sample to clear (~1 min). Carefully pipet and discard the supernatant. 4. Carefully resuspend the beads via pipetting (try to prevent foaming) with equal amount of Bead Binding Buffer (stored at 4°C).5. Place the tube with beads on the Surebeads Magnetic Rack, wait for sample to clear (~1 min), and carefully pipet and discard the supernatant. 6. Carefully resuspend beads via pipetting (try to prevent foaming) with equal amount of Bead Binding Buffer (stored at 4°C).
This protocol is written to use half reactions, which we have found works well for yeast RNA-seq libraries. We suggest piloting half reactions for a small number of samples to determine whether your library yields are sufficient. Reagent amounts can be doubled for full reactions.
?. [mRNA Capture]
1. Transfer 500 ng of RNA to an RNase-free PCR tube, and bring the volume up to 25 μL with RNase-free water.2. Add 25 μL of prepped mRNA Capture Beads to PCR tubes containing total RNA. Thoroughly mix via gently pipetting.3. Place tubes in thermocycler and run:                                Step	                        Temperature	    Duration                                1st mRNA Capture                     65°C          2 min                                Cool                                              20°C	         5 min4. Remove tubes and place on magnetic rack (Diagenode #  A1B04000001) and incubate at room temperature (RT) until sample is clear (~1 min). Use a pipettor to carefully remove supernatant.4. Remove tubes from rack and gently resuspend in 100 μL mRNA Bead Wash Buffer.5. Place tubes on magnetic rack (0.2 mL) and incubate at RT until sample is clear (~1 min). Use a pipettor to carefully remove supernatant.7. Remove tubes from magnetic rack and gently resuspend in 25 μL RNase-free water.8. Place tubes in thermocycler and run:                                 Step	                        Temperature     Duration                                 2nd mRNA Capture                   70°C          2 min                                 Cool	                                      20°C	         5 min9. Add 25 μL Bead Binding Buffer to the tubes. Mix thoroughly via gentle pipetting.10. Incubate tubes at 20°C for 5 min.11. Place tubes on magnetic rack and incubate at RT until sample is clear (~1 min). Use a pipettor carefully remove supernatant.12. Remove tubes from rack and gently resuspend in 100 μL mRNA Bead Wash Buffer.13. Again, place tubes on magnetic rack and incubate at RT until sample is clear (~1 min). Use a pipettor carefully remove supernatant.
The protocol requires 50 ng to 1 μg of total RNA in 25 μL RNase-Free water.We recommend 500 ng of total RNA as a starting point.
A1B04000001
?. [mRNA Elution, Fragmentation, and Priming]
1. Prepare Fragment, Prime, and Elute (FPE) Mastermix.2. Add 11 μL FPE Mastermix to each sample and thoroughly resuspend via pipetting.3. Have a thermocycler pre-incubated to desired temperature (placing samples in a cold thermocycler and ramping up to the temperature can cause over fragmentation,  as samples are exposed to additional heating as the thermocycler gets up to temperature).4. Place ring magnet (Fisher Scientific #A29164) next to thermocycler.5. Place FPE-resuspended sample tubes in a pre-warmed thermocycler and incubate for desired fragment size using a timer (time must be exact. Any longer or shorter will result in differences in fragment sizes).6. Once the incubation is complete, immediately place tubes on ring magnet.7. Using a pipettor, carefully transfer 10 μL supernatant to a fresh RNase-free PCR tube.8. Place tube on ice and proceed immediately to 1st Strand Synthesis.
FPE Mastermix (per sample, if doing more than one sample make 1.05 times the number of samples)    5.5 μL RNase-Free water    5.5 μL Fragment, Prime, and Elute Buffer (2X)Incubation times and temperatures will vary depending on your desired fragment size.    For 100-200 bp, incubate at 94ºC for 8 minutes    For 201-300 bp, incubate at 94ºC for 6 minutes    For 301-400 bp, incubate at 85ºC for 6 minutes
This is a safe stopping point. FPE resuspended samples may be stored at 4ºC ≤ 24 hrs. Freezing samples will damage beads. DO NOT FREEZE.
?. [1st Strand Synthesis]
1. Prepare 1st Strand Synthesis Mastermix.2. Add 5 μL 1st Strand Synthesis Mastermix to the tube containing 10 μL fragmented RNA.3. Keeping tubes on ice, gently mix thoroughly via pipetting.4. Place tubes in a thermocycler and run:                    Step                                Temperature            Duration                    Primer Extension                        25ºC              10 min                    1st Strand Synthesis                  42ºC              15 min                    Enzyme Inactivation                   70ºC              15 min                    HOLD                                               4ºC                   ∞5. Place tubes on ice.6. Proceed immediately to 2nd Strand Synthesis and A-tailing.
1st Strand Synthesis Mastermix (per sample, if doing more than one sample make 1.05 times the number of samples)    5.5 μL 1st Strand Synthesis Buffer    0.5 μL KAPA Script
?. [2nd Strand Synthesis and A-tailing]
1. Prepare 2nd Strand Synthesis and A-tailing Mastermix.2. Add 15 μL 2nd Strand Synthesis Mastermix to the tube containing 15 μL 1st Strand Synthesized samples.3. Place tubes in a thermocycler and run:                    Step                                Temperature            Duration                    2nd Strand Synthesis                 16ºC              30 min                    A-tailing                                        62ºC              10 min                    HOLD                                               4ºC                   ∞5. Place tubes on ice.6. Proceed immediately to Adapter Ligation.
2nd Strand Synthesis Mastermix (per sample, if doing more than one sample make 1.05 times the number of samples)    15.5 μL 1st Strand Synthesis Buffer          1 μL KAPA Script
?. [Adapter Ligation]
1. Dilute Adapters.2. Prepare Adapter Ligation Mastermix.3. Add 2.5 μL diluted adapters to 30 μL 2nd Strand Synthesized samples.4. Keeping tubes on ice, gently mix thoroughly via pipetting.5. Add 22.5 μL Adapter Ligation Mastermix to the 32.5 μL adapter containing 2nd Strand Synthesis samples.6. Keeping tubes on ice, gently mix thoroughly via pipetting.7. Incubate at 20ºC for 15 min.8. Proceed immediately to 1st Post-Ligation Cleanup.
Dilute adapters according to quantity of starting RNA concentration.    Quantity of RNA        Adapter stock concentration    50 ηg - 499 ng                             1.5 μM    500 ηg - 1000ng                             7 μMI stared with 500 ng of total RNA and used 1.5 μM adapter concentration.
Adapter Ligation Mastermix (per sample, if doing more than one sample make 1.05 times the number of samples)        20 μL Ligation Buffer (viscous, pipet slowly)          5 μL  DNA Ligase
?. [1st Post-Ligation Cleanup]
1. Add 35 μL KAPA Pure Beads to 55 μL Adapter ligated samples and mix thoroughly via pipetting.2. Incubate at RT for 5 min.3. Place tubes on ring magnet (Fisher #A29164). Incubate until liquid is clear (~1 min).4. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).5. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip (see "Before Start" in Guidelines & Warnings).6. Incubate at RT for 30 sec.7. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).8. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip.9. Incubate at RT for 30 sec.10. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).11. Using a fresh pipet tip, remove residual ethanol.12. Dry beads at RT for 5 min (over-drying beads may reduce library yield).13. Remove tubes from ring magnet.14. Using a pipettor thoroughly resuspend beads in 25 μL RNase-Free 10 mM Tris-HCl (pH 8.0-8.5).15. Incubate tubes at RT for 2 min.16. Proceed to 2nd Post-ligation Cleanup.
KAPA Pure Beads must be warmed up to RT (~30 min)All ethanol washes will be done on the ring magnet (Fisher #A29164)
This is a safe stopping point.Resuspended beads can be stored at 4ºC for ≤24hrs. DO NOT FREEZE beads, as this will cause dramatic loss of DNA.
?. [2nd Post-ligation Cleanup]
1. Add 17.5 μL PEG/NaCl to 25 μL 1st Post-ligation cleaned samples and mix thoroughly via pipetting.2. Incubate at RT for 5 min.3. Place tubes on ring magnet (Fisher #A29164). Incubate until liquid is clear (~1 min).4. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).5. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip.6. Incubate at RT for 30 sec.7. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).8. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip.9. Incubate at RT for 30 sec.10. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).11. Using a fresh pipet tip, remove residual ethanol.12. Dry beads at RT for 5 min (over-drying beads may reduce library yield).13. Remove tubes from magnet.14. Using a pipettor, thoroughly resuspend beads in 11 μL RNase-Free 10 mM Tris-HCl (pH 8.0-8.5).15. Incubate tubes at RT for 2 min.16. Place tubes on magnetic rack (0.2mL). Incubate until liquid is clear (~1 min).17. Transfer the liquid (10 μL) to a new RNase-Free PCR tube, being careful to not transfer any beads.18. Proceed to Library Amplification.
PEG/NaCl must be warmed up to RT (~30 min)All ethanol washes will be done on the ring magnet (Fisher #A29164).
This is a safe stopping point.Eluted libraries can be stored at 4ºC for ≤1 week or at -20ºC for ≤1 month.
?. [Library Amplification]
1. Prepare Library Amplification Mastermix.2. Add 15 μL Library Amplification Mastermix to 10 μL 2nd Post-ligation cleaned samples. Mix thoroughly via pipetting.3. Place samples in thermocycler and run:                    Step                                Temperature            Duration        Cycles                    Intial Denaturation                     98ºC               45 sec                  1                    Denaturation                               98ºC               15 sec             \                      Annealing                                    60ºC               30 sec                 9                    Extension                                    72ºC               30 sec              /                     Final Extension                          72ºC                 1 min                   1                    HOLD                                              4ºC                   ∞                      4. Proceed immediately to Library Amplification Cleanup.
Library Amplification MAstermix      12.5 μL KAPA HiFi HotStart ReadyMix        2.5 μL  Library Amplifcation Primer MixFor 500 ng starting RNA, 9 cycles worked well. This should be determined empirically.
?. [Libary Amplification Cleanup]
1. Add 25 μL KAPA Pure Beads to 25 μL Library Amplified samples and mix thoroughly via pipetting.2. Incubate at RT for 5 min.3. Place tubes on ring magnet (Fisher #A29164). Incubate until liquid is clear (~1 min).4. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).5. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip6. Incubate at RT for 30 sec.7. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).8. Carefully, dispense 100 μL 80% ethanol into the tube, making sure to not touch the tube with the pipet tip9. Incubate at RT for 30 sec.10. Carefully, without touching the beads, use a pipettor to remove the supernatant (using Avant #AH200-H - skinnier tips than normal barrier tips).11. Using a fresh pipet tip, remove residual ethanol.12. Dry beads at RT for 5 min (over-drying beads may reduce library yield).13. Remove tubes from ring magnet.14. Using a pipettor thoroughly resuspend beads in 11 μL RNase-Free 10 mM Tris-HCl (pH 8.0-8.5).15. Incubate tubes at RT for 2 min.16. Place tubes on magnetic rack (0.2 mL). Incubate until liquid is clear (~1 min).17. Transfer clear (no beads) 10 μL to a new RNase-Free PCR tube.
KAPA Pure Beads must be warmed up to RT (~30 min)All ethanol washes will be done on the ring magnet (Fisher #A29164).
RNA-Seq library is completed. Quantify and check quality using an Agilent TapeStation or BioAnalyzer.