characteristics.text: SAMN00117444
characteristics.characteristic: External Id
characteristics.text: Michigan State University
characteristics.characteristic: INSDC center name
characteristics.text: 2010-11-08T09:37:16.060Z
characteristics.characteristic: INSDC first public
characteristics.text: 2013-06-12T09:08:11.903Z
characteristics.characteristic: INSDC last update
characteristics.text: SRS118977
characteristics.characteristic: INSDC secondary accession
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: SRS118977
characteristics.characteristic: SRA accession
characteristics.text: Seeds of Momordica charantia collected at 18 days after pollination. Independent male flowers were used for hand pollination of female flowers. Embryos were dissected from seeds of fruits collected at specific DAP and frozen immediately in liquid nitrogen. Embryos were stored at -80°C until use in RNA isolation. Total RNA was extracted from developing bitter melon seeds was ground to a fine powder in liquid nitrogen using Trizol reagent (Invitrogen) according too the manufacturer’s protocol.mRNA were purified from 1 mg of total RNA by two passes through oligo-dT cellulose columns by use of the Illustra mRNA purification kit (GE Healthcare). cDNA libraries were constructed using SMART PCR cDNA synthesis kit (Clontech). First-strand cDNA was synthesized with 150 ng mRNA in a volume of 10 ml using the provided SMART II primer, a modified CDS III/ 3’ cDNA synthesis primer (5’-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCGACATGTTTTGTTTTTTTTTCTTTTTTTTTTVN-3’) and Superscript II reverse transcriptase (Invitrogen). Double stranded cDNA was prepared by PCR (18 cycles) using 2 ml of the first-strand reaction in a 50 ml reaction volume. Following Proteinase K treatment, four PCR reactions were pooled before SfiI digestion and size fractionated on the provided CHROMA SPIN-400 column. Only fractions containing fragments larger than 500 bp were collected, precipitated, and resuspended in TE buffer. Library normalization of this cDNA was conducted by use of Trimmer-Direct cDNA normalization kit (Evrogen). Briefly, four 250 ng aliquots of cDNA were hybridized at 98 °C for 2 min followed by 68 °C for 5 h. The hybridized cDNAs were then treated with 0, 0.25, 0.5, and 1 ml duplex-specific nuclease (DSN), respectively, before stop with the DSN stop buffer. cDNA (1 μl) from each aliquot was subjected to PCR amplification. Based on the results from the sample lacking DSN, the cycle number (9+2 cycles) was determined for the first round amplification of DSN treated samples. After examination of the cDNAs on the agarose gel, the selected aliquot of cDNAs were then diluted 10 times and subjected to a second round of PCR using 2 ml in 100 ml reaction (12 cycles). The amplified cDNA pool was then treated with proteinase K, fractionated, and precipitated for the non-normalized cDNA library construction. For the pilot study, cDNA PCR fragments were digested with SfiI enzyme and cloned into SfiIA and SfiIB sites of pDNR-LIB vector (Clontech).
characteristics.characteristic: description
characteristics.text: Momordica charantia
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_3673
characteristics.characteristic: organism
characteristics.text: Normalized cDNA library from Momordica charantia seeds
characteristics.characteristic: title