Directional-RNAseq Prep

To make strand-specific mRNA libraries, we adapted the method of Parkhomchuk et al. (Nucl. Acids Res., 2009, doi: 10.1093/nar/gkp596). In a comparison of several different strand-specific methods, Levin et al. (Nat Methods 2010, 7:709-715) found the dUTP method to be among the most reliable tested. Our method has been optimized for the Illumina TruSeq RNA sample preparation kit, but it can likely be used for any library construction method that procedes via polymerase-mediated double-stranded cDNA/second strand synthesis.

Materials required for making Strand-Specific mRNAseq Illumina libraries via dUTP

• 48 well plate or RNase-free, non-sticky 1.5 mL tubes

• Illumina TruSeq RNA Sample preparation kit (Illumina #RS930-2001; manual 15008136 Rev. A, November 2010)

• Superscript III first strand synthesis kit (Invitrogen 18080-051)

• NEBNext Second Strand Synthesis Buffer, dNTP-Free (NEB B6117S)

• NEBNext mRNA Second Strand Synthesis enzyme mix (NEB E6111S)

• 2 mM dNTP/ 4 mM dUTP Mix (Fermentas R0251)

• USER (uracil-specific excision reagent) Enzyme mix (NEB M5505S)

• 5M NH4OAc; Glycoblue (Ambion) or similar coprecipitant; 100% Ethanol; RNAse-free water

These steps replace or are added to the Illumina TruSeq RNA Sample preparation guide

---- First Strand Synthesis (page 44) ---

1. Perform First Strand Synthesis (FSS) reaction as directed.

2. When the FSS reaction is complete, precipitate RNA with ethanol and ammonium acetate. Per 50 ul FSS reaction, mix:

    •	5 ul 5M NH4OAc (ammonium is used because it does not precipitate nucleotides).
    •	2 ul GlycoBlue (or similar co-precipitant)
    •	110 ul 100% Ethanol

3. Incubate 1 hour to overnight at -80°C

4. Centrifuge 30 min x 20,000g, 4°C

5. Remove supernatant; add 200 ul of ice-cold 70% EtOH

6. Centrifuge 5 min x 20,000g, 4°C

7. Remove supernatant; air dry 10 minutes.

---- Resuspend pellet in FSS buffer w/o dNTPs or RT (new step) ---

1. Per sample, mix the following reagents from the Superscript III kit together in a master mix:

    •	2 ul 10X RT buffer 
    •	4 ul 25 mM MgCl2
    •	2 ul 0.1 M DTT
    •	1 ul random hexamers (50 ng/ul)
    •	49 ul dH2O 

2. Add 58 ul of the master mix to each pellet and resuspend by pipetting gently.

---- Perform Second Strand Synthesis (SSS) with dUTP/dNTP mix (new step) ---

1. Per sample, mix the following into a master mix:

    •	8 ul of 10X dNTP-FREE Second Strand buffer (NEB B6117S)
    •	10 ul dNTP/dUTP mixt
    •	4 ul Second Strand enzyme mix (NEB E6111S)

2. Pipette 22 ul of the master mix into each resuspended RNA to give a final volume 80 ul.

3. Incubate 2.5 hours at 16°C.

---- Clean the SSS product with AMPure, as directed (page 48) ---

---- Continue protocol as directed through the AMPure cleaning step following adapter ligation (page 48) ---

---- Prior to PCR enrichment, degrade the dUTP in the coding strand (new step) ---

1. Per sample, add 1 ul of USER enzyme mixture.

2. Incubate at 37°C for 15 minutes, then place on ice

---- Continue to PCR enrichment step (page 62) ---

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