title: GSM4504304: 222; Rattus norvegicus; RNA-Seq
library_construction_protocol: Samples from the colon were homogenized in TRIzol reagent by ULTRA-TURRAX T-18 (IKA) instrument. 0,1 ml of chloroform (Sigma-Aldrich) was added to 0.3 ml homogenized sample with vigorous vortexing. Samples were centrifuged at 13000 rpm for 10 minutes. Total RNA was than extracted from the upper aqueous phase with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. RNA-Seq was performed by SOLiD total RNA-Seq Kit (Life Technologies), according to the manufacturer's instructions and sequenced on SOLiD 5500xl instrument using the 50-base sequencing chemistry. For this, 5µg of RNA was DNaseI treated and the ribosomal RNA depleted using RiboMinus Eucaryote kit for RNA-Seq and RiboMinus Concentration Module all three from Thermo Fisher Scientific, Waltham, MA USA. The leftover was fragmented using RNaseIII (part of the kit), the 50-200 nt fraction size-selected, sequencing adaptors ligated and the templates reverse transcribed using ArrayScript reverse transcriptase (part of the kit). The cDNA library was purified with Qiagen MinElute PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) and size-selected on a 6% TBE-Urea denaturing polyacrylamide gel (Thermo Fisher Scientific, Waltham, MA USA). The 150-250 nt cDNA fraction was amplified using AmpliTaq polymerase (Thermo Fisher Scientific, Waltham, MA USA) and purified by AmPureXP Beads (Agencourt, Beckman Coulter GmbH Krefeld, Deutschland). Concentration of each library was determined using the SOLiID Library TaqMan Quantitation Kit (Thermo Fisher Scientific, Waltham, MA USA). Each library was clonally amplified on SOLiD P1 DNA Beads by emulsion PCR (ePCR) (Thermo Fisher Scientific, Waltham, MA USA). Emulsions were broken with butanol (Thermo Fisher Scientific, Waltham, MA USA) and ePCR beads enriched for template-positive beads by hybridization with magnetic enrichment beads (Thermo Fisher Scientific, Waltham, MA USA). Template-enriched beads were extended at the 3' end in the presence of terminal transferase and 3' bead linker. Beads with the clonally amplified DNA were deposited onto SOLiD flowchip (Thermo Fisher Scientific, Waltham, MA USA) and sequenced on SOLiD 5500xl System (Thermo Fisher Scientific, Waltham, MA USA).
attributes.tag: GEO Accession
attributes.value: GSM4504304