title: GSM1303830: 108hpf_wt; Danio rerio; RNA-Seq
library_construction_protocol: Genomic DNA and total RNA were extracted concurrently form single embryos by acid guanidinium thiocyanate-phenol-chloroform extraction. Genomic DNA extraction and genotyping of individual embryos was completed first, and aqueous phases of all homozygous wildtype and mutant larvae, respectively, were pooled prior to RNA precipiation with isopropanol. For RNA sequencing, RNA was extracted from approx 60 pooled genotyped larvae and subsequently enriched for polyA RNA by two successive rounds of polyA selection with the MicroPoly(A)Purist Kit (Ambion). Libraries for deep sequencing were generated using the SOLiD™ Whole Transcriptome Analysis Kit with protocol Part Number 4409491 Rev F, 08/2009. 8μl of poly(A) RNA were was fragmented by incubating with RNase III for 10min at 37°C and immediately purified on Qiagen RNeasy columns. Samples were eluted in 50μl of nuclease-free water and the columns incubated for 10min before the final centrifugation. Samples were concentrated to a volume of ~5μl by centrifuging under vacuum at 40°C. 1μl of each sample was analysed on the Agilent Bioanalyzer (Agilent) using an RNA Nano 6000 chip (Agilent) to assess quantity and size distribution of the fragmented RNA. The fragmented RNA was hybridised and ligated to Adaptor Mix A to generate templates for SOLiD System sequencing from the 5’ end of the sense strand. After o/n ligation, samples were reverse-transcribed for 30min at 42°C and the obtained cDNA purified using Qiagen inElute PCR Purification Kit. 100-200bp fragments were size selected by excision from a 6% polyacrylamide, 8M urea-TBE gels. cDNA in gel slices was subjected to 18 cycles of amplification and purified using PureLink PCR Micro columns (Invitrogen). The majority of cDNAs in the library was between 150 and 250bp in size and less than 2-8% of cDNAs fell in the 25-150bp size range as per protocol requirements. The finished cDNA libraries were used for attachment to beads by emulsion PCR.
attributes.tag: GEO Accession
attributes.value: GSM1303830