Directional-RNAseq Prep

Directional dUTP-based RNAseq Libraries using the Illumina TruSeq RNA sample preparation kit (catalog RS930-2001, manual 15008136 Rev. A, November 2010) September, 2011 -- IN PROGRESS! These steps either replace or are added to the Illumina TruSeq RNA Sample preparation guide. The can be used with any other preparation method that uses independent steps for First Strand Synthesis (FSS) and Second Strand Synthesis (SSS).

(1) Perform FSS reaction as directed (e.g., pg. 44 of the Illumina TruSeq RNA Sample Preparation Guide 15008136 Rev A). (2) After the FSS reaction is complete, use ethanol and ammonium acetate to precipitate the RNA and leave unincorporated nucleotides in solution. Per 50 ul FSS reaction, mix: • 5 ul 5M NH4OAc (ammonium is used instead of sodium 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.

(3) 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.

(4) 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) 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 (page 61).

(7) Prior 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) Proceed to the PCR Enrichment step (page 62).