alias: GSM1564951
title: GSM1564951: 12 hour post bisection, rep1; Hydra vulgaris; RNA-Seq
library_construction_protocol: 30 μl 3M sodium acetate and 400 ul Tris-EDTA saturated phenol were added to each sample and mixed. Then 80 μl of chloroform was added to each sample and placed on ice for 15 minutes. Subsequently, the samples were centrifuged for 20 minute at 14,000 RPM. Subsequent centrifugation steps were also carried out at this speed. The aqueous phase of each sample was transferred to a fresh tube and an equal volume of isopropanol added. The samples were stored at -20°C for 2 hours and then centrifuged at 4°C for 15 minutes. After removal of the fluid, the pellet was washed in 70% ethanol. Each pellet was dissolved in 30-100 μl DEPC-treated water and an equal volume of 5M LiCl added. The samples were stored on ice at 4°C overnight. Subsequently the samples were centrifuged for 30 minutes and the LiCl removed. The pellets were washed twice with 70% ethanol and air-dried. Finally, each pellet was dissolved in 10 μl DEPC treated water and subsequently used for poly(A) selection or stored at -80°C. The total RNA integrity was checked using Bioanalyzer. All samples had RIN numbers greater than 9.0. The isolated total RNA was enriched for mRNA using two rounds of poly(A) selection with a Dynabeads mRNA DIRECT Kit (Invitrogen). Purified mRNA was fragmented with hydrolysis buffer containing magnesium ions, which was then used to prepare double stranded cDNA using Invitrogen’s SuperScript Double-stranded cDNA preparation kit. During second strand synthesis, dUNTP (Applied Biosystems) was used to label the second-strand. The Illumina sequencing library construction protocol of Myers Lab ChIP-seq protocol v.2011 was used for the preparation of multiplexed libraries. The samples were treated with Uracil N-glycosylase (Applied Biosystems) before the PCR amplification step to create the strand-specific RNA-seq libraries. 13-15 cycles of PCR were used depending on the initial amount of cDNA used for each sample. Libraries were sequenced as 50 bp multiplexed Illumina single-end reads on a HiSeq2500.
identifiers.namespace: GEO
identifiers.id: GSM1564951
attributes.tag: GEO Accession
attributes.value: GSM1564951