PROTOCOL FOR LIBRARY PREPARATION using KAPA RNA Hyper Prep Kit w/RiboErase

Procedure

400 ng DNase-treated RNA and a total of 9 cycles of PCR amplification of the library. The libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Kapa Biosystems). We followed the step-by-step protocol from the manufacturer (see Reference 2), attached using 400 ng DNase-treated RNA and a total of 9 cycles of PCR amplification of the library.

1. Ensure samples meet following requirements
   

   RNA RIN >6
   RNA in 10 μl Elution Buffer
   25 ng < RNA < 1 μg
   # Samples: 12

   
   
2. Oligo Hybridization and rRNA Depletion
   

   Hybridization MM	1X	12
   KAPA Hyb Buffer	4	52.8
   KAPA Hyb Oligo	4	52.8
   Water	2	26.4	<-- Keep MM at RT
   RNA	10	--
   Total Volume	20

   **Add 10 μl Hyb MM to RNA once Depletion MM is made.
   
   

   rRNA Depletion MM	1X	12
   KAPA Depletion Buffer	3	39.6
   KAPA RNase H	2	26.4	<-- Keep MM at RT
   RNA	20	--
   Total Volume	25

   **Add 5 μl MM to RNA once cycler at 45 °C. Pipette up/down.
   
   

   	Thermal Cycler Conditions	Temp	Time
   1	Hybridization	95 °C	2 min
   2	Ramp down to 45 °C at -0.1 °C/s
   3	Pause	45 °C	-
   4	Depletion	45 °C	30 min
   5	Hold	4 °C	-

   
   
3. rRNA Depletion Cleanup
   **Equilibrate beads to RT before use.
   **Make 80% EtOH.
   
   

   2.2X KAPA Pure Bead	1 Rxn (μl)
   Ribosomal-depleted RNA	55
   KAPA Pure Beads	55
   Total Volume	80

   
   
   1. Mix samples by pipetting and incubate at RT for 5 min.
   2. Place tubes on magnetic stand and discard 75 μl supernatant.
   3. Add 200 μl 80% EtOH and incubate for 30 s.
      80% EtOH for 12 samples:
      4.8 ml 100% EtOH
      1.2 ml dH₂O
      6.0 ml Total
   4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
   5. Remove EtOH and dry for 3-5 min.
      

   **DO NOT OVERDRY.
   **Proceed immediately to next step.

4. DNase Digestion
   

   DNase Digestion MM	1X	12
   KAPA DNase Buffer	2.2	29.0
   KAPA DNase	2.0	26.4
   Water	17.8	235.0	<-- Keep MM at RT
   Total Volume	22

   **Add 22 μl MM to each tube, resuspend beads.
   
   
   1. Incubate beads at RT for 3 min.
   2. Place on magnetic stand and transfer 20 μl of supernatant to new tube.
      **Discard remaining beads.
   3. Incubate tubes at 37 °C for 30 min and hold at 4 °C.
      
      
5. DNase Digestion Cleanup
   **Equilibrate beads to RT before use.
   **Make 80% EtOH.
   
   

   2.2X KAPA Pure Bead	1x
   DNase-treated RNA	20
   KAPA Pure Beads44	44
   Total Volume	64

   
   
   1. Mix samples by pipetting and incubate at RT for 5 min.
   2. Place tubes on magnetic stand and discard 60 μl supernatant.
   3. Add 200 μl 80% EtOH and incubate for 30 s.
   4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
   5. Remove EtOH and dry for 3-5 min.
      

   **DO NOT OVERDRY.
   **Proceed immediately to next step.

6. RNA Elution, Fragmentation and Priming
   

   1X Fragment Buffer	1X	12
   RNase-free Water	11	145.2
   Fragment, Prime Elute Buffer	11	145.2	<-- Keep at RT
   Total Volume	22

   
   **Add 22 μl to each tube and resuspend beads.
   
   1. Incubate at RT for 3 min and place on magnetic stand.
   2. Transfer 20 μl of supernatant to new tube.
      **Discard tube with beads.
      **Safe stopping point: Store at -20 °C for 24 h.
      
      

   Input RNA Type	Desired Size (bp)	Fragmentation
   Intact	100-200	8 min at 94 °C
   	200-300	6 min at 94 °C
   	300-400	6 min at 85 °C
   Partially Degraded	100-300	1-6 min at 85 °C
   Degraded	100-200	1 min at 65 °C

   Place tubes on ice and proceed to next step.
   
   
7. 1st Stand Synthesis
   

   1st Strand Synthesis	1X	12
   1st Strand Synthesis Buffer	31	372
   KAPA Script	2	24	<-- Keep on ice
   1st Strand Synthesis product	30	--
   Total Volume	60

   **Add 10 μl MM to each RNA sample.
   
   Mix samples with pipetting and incubate with following protocol:
   

   	Thermal Cycler Conditions:	Temp	Time
   1	Primer extension	25 °C	10 min
   2	1st Strand Synthesis	42 °C	15 min
   3	Enzyme inactivation	70 °C	15 min
   4	Hold	4 °C	-

   **Place tube on ice and proceed immediately to next step.
   
   
8. 2nd Strand Synthesis
   

   2nd Strand Synthesis	1x	12
   2nd Strand Marking Buffer	11	132
   2nd Strand Synthesis & A tail Mix	1	12	<-- Keep on ice
   Fragmented, Primed RNA	20
   Total Volume	30

   **Add 30 μl MM to each sample.
   
   Mix samples with pipetting and incubate with following protocol:
   

   	Thermal Cycler Conditions:	Temp	Time
   1	2nd Strand Synthesis	16 °C	30 min
   2	A Tailing	62 °C	10 min
   3	Hold	4 °C	-

   **Place tube on ice and proceed immediately to next step.
   
   
9. Adapter Ligation
   

   Adapter Ligation	1X	12
   Ligation Buffer	40	480
   DNA Ligase	40	120	<-- Keep on ice
   Adapter stock (1.5 μM)	5	--
   2nd Strand Synthesis product	60	--
   Total Volume	110

   **Add 45 μl MM to each sample. **Add adapter first then add mastermix.
   
   
   1. Mix samples on ice and incubate at 20 °C for 15 min.
   2. Proceed immediately to next step.
      
      
10. 1st Post-Ligation Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.
    
    

    0.63X KAPA Pure Bead	1X
    Adapter Ligated DNA	110
    KAPA Pure Beads	70
    Total Volume	180

    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 175 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
       **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 50 μl 10 mM Tris-HCl, pH 8.0.
    7. Incubate beads at RT for 2 min.
       **Safe stopping point: Store at 4 °C for 24 h.
       
       
11. 2nd Post-Ligation Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.
    
    

    0.7X KAPA Pure Bead	1X
    Beads w/ Adapter-ligated DNA	50
    20% PEG 8000/2.5 M NaCl Solution	35	<-- Equilibrate to RT, mix before use
    Total Volume	85

    
    
    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 80 μl supernatant.
       
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
       **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 22.5 μl 10 mM Tris-HCl, pH 8.0.
    7. Incubate beads at RT for 2 min.
    8. Place on magnetic stand and transfer 20 μl supernatant to new tube.
       **Safe stopping point: Store at 4 °C for 1 wk or -20 °C for 1 mo.
       
       
12. Library Amplification
    

    Library Amplification	1X	12
    KAPA HiFi HotStart Mix	25	330	<-- Fully thaw and mix before use
    Library Amp Primer Mix	5	66	<-- Keep on ice
    Purified, Adapter-ligated DNA	20	--
    Total Volume	50

    **Add 30 μl MM to each sample.
    
    

    	Thermal Cycler Conditions:	Temp	Time
    1	Initial Denaturation	98	45 s
    2	Denaturation	98	15 s
    3	Annealing	60	30 s
    4	Extension	72	30 s
    5	Extension	8 more times	<-- Depends on quantity of starting material
    6	Final Extension	72	1 min
    7	Hold	4	-

    Proceed immediately to next step.
    
    
13. Library Amplification Cleanup
    **Equilibrate beads to RT before use.
    **Make 80% EtOH.
    
    

    1X KAPA Pure Bead	1X
    Amplified library DNA	50
    KAPA Pure Beads	35
    Total Volume	85

    
    
    1. Mix samples by pipetting and incubate at RT for 10 min.
    2. Place tubes on magnetic stand and discard 82 μl supernatant.
    3. Add 200 μl 80% EtOH and incubate for 30 s.
    4. Remove EtOH, add 200 μl 80% EtOH, incubate for 30 s.
    5. Remove EtOH and dry for 3-5 min.
       **DO NOT OVERDRY.
    6. Remove beads from stand and resuspend beads in 22 μl 10 mM Tris-HCl, pH 8.
    7. Incubate beads at RT for 2 min.
    8. Place on magnetic stand and transfer 20 μl supernatant to new tube.
    9. Submit samples for QC/Seq.
    10. Store at 4 °C for < 1 week, or at -20 °C.

References

1. Whipple, A. J., Jacobs, H. N., Breton-Provencher, V., Sur, M. and Sharp, P. A. (2019). Imprinted maternally-expressed microRNAs antagonize paternally-driven gene programs in neurons. bioRxiv: 717868.
2. Kapa Biosystems. (2018). KAPA RNA HyperPrep Workflow: Recommendations and Expectations for RNA-sequencing Using Degraded Inputs. Kapa Biosystems. Wilmington, MA, USA, and Cape Town, South Africa.