title: GSM1146899: ExoCAGE of BMDCs 0hrs post LPS stimulation; Mus musculus; RNA-Seq
library_construction_protocol: 50ng-1ug of total RNA is polyA selected using Oligo-dT beads (Invitrogen) and then fragmented using Ambion Fragmentation buffer using incubation time of 1 min 50sec at 70C. Extractions for ExoCAGE and 3' RNASeq are carried out as described in the paper. 50ng-1ug of total RNA is polyA selected using Oligo-dT beads (Invitrogen) and then fragmented using Ambion Fragmentation buffer using incubation time of 1 min 50sec at 70C.   ExoCAGE: Fragmented RNA is cleaned-up using 2.5X volume on SPRI beads (Agencourt), and incubated with Terminator Exonuclease (Epicentre) for 90 minutes. Reaction mixture is then cleaned up with 2.5X of SPRI beads and then processed the same way as above starting with dephosphorylation and lygation steps. 3' RNA-Seq: Fragmented RNA is cleaned-up using 2.5X volume on SPRI beads (Agencourt) and then subjected to second polyA enrichment step. Specifically, we used cDNA synthesis with custom oligo-dT priming (CCTACACGACGCTCTTCCGATCT-T15) for mouse dendritic cells. Reaction mixture is then processed in the same way as in Full-Length protocol starting with hybrid degradation and so on.