modENCODE D. melanogaster poly(A) site-seq
Drosophila melanogaster poly(A) Site-Seq
RNA sequencing libraries specific for polyadenylation sites were prepared as follows. RNA samples, from dissected heads of males and mated females at 20 days post-eclosion, were used to produce two âpolyA-seqâ libraries. Total RNA (2 Âµg) was fragmented in 1X RNA Fragmentation Reagent (Life Technologies) in 10 Âµl at 65Â°C for 5 minutes. The reaction was stopped by addition of 1 Âµl of reaction stop buffer (Life Technologies) and cooled on ice. The fragmented RNA sample was used, without precipitation, as the starting material for the library construction protocol and kit described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide (Rev. D, September 2012), with the following modifications. At the second round of poly(A)+ RNA selection, the bound RNA was eluted with addition of 13.5 Âµl of nuclease-free water and heating to 65Â° for 5 minutes. The eluted RNA was removed from the beads in 11 Âµl, and 1 Âµl of a custom anchored oligo-dT primer (20 Âµg/Âµl; 5â-NGCAGCAT(20)VN-3â) and 5 Âµl of 5X Superscript II Buffer (Life Technologies) were added. The sample was heated to 42Â° for 2 minutes to anneal the primer, then cooled on ice. The annealed sample was used to prepare a sequencing library following the remaining steps in the Illumina protocol from first-strand cDNA synthesis to the end. Libraries were sequenced on the Illumina HiSeq platform to produced paired-end reads (2 x 100 nt) following standard protocols.
These data were produced for the modENCODE project (http://www.modencode.org/). For a description of the protocols and other experimental details please refer to the data distribution site http://www.modencode.org/Celniker.shtml. In accordance with NHGRI guidelines for community resource projects (http://www.genome.gov/), there is no restriction on the use or analysis of these data. However, the end-user is asked to respect a nine-month moratorium (from the public submission date) before submitting manuscripts describing analysis of these data. If the data producer has published any description of these data before the nine-month period, the moratorium ceases to apply and this accession page will be updated to note the appropriate citation.