Design: Ten unrelated great tit fledglings were sampled from across the Wytham Woods population and brain, heart, kidney, liver, muscle, pancreas, skin and testes/ovaries dissected from each individual at between three and 23 minutes after death. Each chick was sexed at sampling and sex was later confirmed molecularly with the Z-002A marker. Tissues were stored in RNALater (Ambion) to prevent RNA degradation. RNA was extracted using the TRIzol® (Invitrogen) method. RNA quantity and degradation was tested using a Nanodrop (Agilent) with each tissue yielding a mean (SE) of 44.4 (7.5) ug of RNA with mean (SE) RNA integrity number (RIN) of 8.37 (0.21). For each tissue 1ug of RNA from each bird was pooled, i.e. to make 8 pools, each containing 10µg of RNA. Complementary DNA (cDNA) was synthesised by Evrogen (Moscow, Russia) from the eight tissue-specific pools using SMART kits. cDNA was normalised, also by Evrogen. cDNA sequencing was performed by the Liverpool Advanced Genomics Facility, with 3ug of normalised cDNA sequenced on a 454 FLX Titanium Genome Sequencer. Each tissue was sequenced on half a plate, i.e. four plates were sequenced in total. tissue: number of sequences, total base pairs sequenced, mean sequence length, maximum sequence length brain: 674719, 209572185, 311, 1796 heart: 408547, 112462495, 275, 1863 kidney: 655004, 198351347, 303, 1256 liver: 514017, 157069646, 306, 1005 muscle: 566729, 178678017, 315, 971 pancreas: 532017, 174131123, 327, 1726 skin: 573505, 154523708 269, 2028 testes: 663036, 199038872, 300, 1737 all tissues: 4587574, 1383827393, 302, 2028
Submitted by: UNIVERSITY OF SHEFFIELD
Study:
Characterisation of the transcriptome of a wild great tit Parus major population by next generation sequencingshow Abstracthide AbstractRNA was extracted from eight tissues of wild great tit fledglings, and a normalised cDNA library generated and sequenced. Approximately 4.6 million sequences and 1.4 billion bases of DNA were generated and assembled into 95,979 contigs. Over 90% of contigs aligned with the zebra finch (Taeniopygia guttata) genome, and one third aligned with known Taeniopygia guttata (zebra finch) and Gallus gallus (chicken) transcripts. More than 35,000 single nucleotide polymorphisms and 10,000 microsatellite repeats were identified.
Sample:
Parus major transcriptome sequencingLibrary:
Name: tissues
Instrument: 454 GS FLX Titanium
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Spot descriptor:
5 forward
Runs:
8 runs, 4.6M spots, 2.6G bases, 5.4Gb