Directional-RNAseq Prep

The modifications we adapted are those from Parkhomchuk et al. (Nucl. Acids Res., 2009, doi: 10.1093/nar/gkp596). This was determined by Levin et al. (Nat Methods 2010, 7:709-715) to be one of the most reliable strand-specific sequencing method tested. While our method is optimized for the Illumina TruSeq RNA sample preparation kit, 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

• Sera-Mag Oligo(dT)₁₄ beads, Seradyn #2815-2103-010150 (Now from Thermo-Forma; 20 µL per sample)

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

• Bead Washing Buffer (10mM Tris-HCl PH 7.5, 0.15M LiCl, 1mM EDTA; user supplied, 700 µL per sample)

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

(1) Page 44: Perform Fist Strand Synthesis (FSS) reaction as directed.

• 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

o incubate 1 hour to overnight at -80°C

o centrifuge 30 min x 20,000g, 4°C

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

o centrifuge 5 min x 20,000g, 4°C

o Remove supernatant; air dry 10 minutes.

(2) New StepItalic text: Resuspend pellet in first strand synthesis buffer w/o dNTPs and RT. Per sample, mix:

• 2 ul10X RT buffer (superscript kit)

• 4 ul 25 mM MgCl2 (superscript kit)

• 2 ul 0.1 M DTT (superscript kit)

• 1 ul random hexamers (superscript kit)

• 49 ul dH2O (superscript kit)

o mix well; add 58 ul to each pellet and resuspend by pipetting gently. The final volume should be 58 ul.

(3) New StepItalic text: Perform Second Strand Synthesis using the following method. Per sample, mix:

• 8 ul of 10X Second Strand buffer mix w/o dNTPs (NEB buffer)

• 10 ul dUTP/dNTP mixture

• 4 ul second strand enzyme mix (NEB)

o mix well. Pipette 22 ul to each resuspended RNA to give a final volume 80 ul.

o Incubate 2.5 hours at 16C.

(5) Page 48: Clean the SSS product with AMPure, as directed in the TruSeq kit.

(6) Continue all steps as directed through the ligation step. After adapter-ligated samples are purified with AMPure, elute in 22.5 ul of Resuspension Buffer (10 mM Tris-HCl) and transfer 20 ul to a new tube or plate, as directed.

(7) New Step: Italic textPrior to PCR Enrichment, add 1 ul of USER mixture to each 20 ul sample. Incubate at 37C for 15 minutes, then place on ice

(8) Page 62: Proceed to the PCR Enrichment step.