cell type: Bone Marrow-derived Dendritic Cell (BMDC)

Cells were cultured and stimulated with LPS as previously described (Amit et. al 2009)

We used the SMARTer Ultra Low RNA Kit (Clontech, Mountain View, CA) to prepare amplified cDNA. We added 1   l of 12   M 3' SMART primer (5'   AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N (N = A, C, G, or T; N-1 = A, G, or C)), 1   l of H2O, and 2.5   l of Reaction Buffer onto the RNA capture beads. We mixed them well by pipetting, heated the mixture at 72  C for 3 minutes and placed it on ice. First-strand cDNA was synthesized with this RNA primer mix by adding 2   l of 5x first-strand buffer, 0.25   l of 100mM DTT, 1   l of 10 mM dNTPs, 1   l of 12   M SMARTer II A Oligo (5'   AAGCAGTGGTATCAACGCAGAGTACXXXXX (X = undisclosed base in the proprietary SMARTer oligo sequence)), 100 U SMARTScribe RT, and 10 U RNase Inhibitor in a total volume of 10   l and incubating at 42  C for 90 minutes followed by 10 minutes at 70  C. We purified the first strand cDNA by adding 25   l of room temperature AMPure XP SPRI beads (Beckman Coulter Genomics, Danvers, MA), mixing well by pipetting, incubating at room temperature for 8 minutes. We removed the supernatant from the beads after a good separation was established. We carried out all of the above steps in a PCR product   free clean room. We amplified the cDNA by adding 5   l of 10x Advantage 2 PCR Buffer, 2   l of 10 mM dNTPs, 2   l of 12   M IS PCR primer (5'    AAGCAGTGGTATCAACGCAGAGT), 2   l of 50x Advantage 2 Polymerase Mix, and 39   l H2O in a total volume of 50   l. We performed the PCR at 95  C for 1 minute, followed by 21 cycles of 15 seconds at 95  C, 30 seconds at 65  C and 6 minutes at 68  C, followed by another 10 minutes at 72  C for final extension. We purified the amplified cDNA by adding 90   l of AMPure XP SPRI beads and washing with 80% ethanol.

We created Illumina sequencing libraries from this amplified cDNA using standard protocols.

We added the purification buffer (Clontech) to the amplified cDNA to make a total volume of 76   l. We sheared the cDNA in a 100   l tube with 10% Duty Cycle, 5% Intensity and 200 Cycles/Burst for 5 minutes in the frequency sweeping mode (Covaris S2 machine, Woburn, MA). We purified the sheared cDNA with 2.2 volumes AMPure XP SPRI beads.