title: GSM1940798: AlNc14_4dpi_biorep1_lib1; Arabidopsis thaliana; OTHER
library_construction_protocol: Total RNA was extracted using the TRI reagent (Sigma) and 1-Bromo-3-chloropropane (Sigma), as per manufacturer’s guidelines. RNA was precipitated with half volume of isopropanol and half volume of high salt precipitation buffer (0.8 M sodium citrate and 1.2 M sodium chloride). RNA samples were treated with DNaseI (Roche) according to the manufacturer’s recommendation and phenol/chloroform extracted and ethanol precipitated. [EXPRSS] Typically 3 µg of total RNA was used to generate first strand cDNA using a oligo(dT) primer comprising P7 sequence of Ilumina flowcell. Double strand cDNA is synthesized as described previously [Okayama & Berg, Molecular and cellular biology 1982, 2:161-170]. Purified cDNA is subjected to Covaris shearing to a target size of 200bp (parameters: Intensity – 5, Duty cycle – 20%, Cycles/Burst – 200, Duration – 60seconds). End repairing and A-tailing of sheared cDNA is carried out as described by Illumina. Y-shaped adapters are ligated to A-tailed DNA and subjected to size selection on 1X TAE agarose gel. The gel-extracted library is PCR enriched and quantified using qPCR with previously sequenced similar size range Illumina library
identifiers.namespace: GEO
identifiers.id: GSM1940798