GSM1244719: Fourth instar Culex quinquefasciatus Time 0 untreated; Culex quinquefasciatus; RNA-Seq
A total of 200 larvae were pooled for each sample, homogenized in quanidinium isothiocyanate buffer and extracted using the hot acid phenol extraction method as outlined by Chomczynski and Sacchi (1987). RNA was extracted over 1:1 acid phenol twice, precipitated over two volumes of 100% ethanol, then resuspended in buffer and treated with Proteinase K to remove remaining protein, re-extracted over 1:1 phenol:chloroform, extracted 1x over chloroform and precipitated over two volumes of ethanol and 0.3M sodium acetate. From this, a total of 30 micrograms of RNA was used for DNase I treatment using the Ambion DNA-Free kit to remove residual DNA. DNA-free RNA was then re-extracted over 1:1 phenol:chloroform followed by final extraction over chloroform, precipitation over two volumes of 100% ethanol and 0.3M sodium acetate. The precipitated pellet was washed two times over 80% ethanol, resuspended in sterile deionized water and sent for sequencing. Paired end libraries were prepared according to Illumina's instructions accompanying the mRNA Seq 8-sample kit by the Hudson Alpha Institute of Biotechnology, Huntsville, AL. Libraries were then sequenced on the Illumina HiSeq 2000 following the manufacturer's protocols. Lengths of 51 nucleotides were sequenced for each of the paired ends and the average insert size was 200 nucleotides.
GEO Accession
GSM1244719