Trachemys scripta embryonic transcriptome - SRA

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SRX121294: Trachemys scripta embryonic transcriptome
1 ILLUMINA (Illumina HiSeq 2000) run: 188.7M spots, 18.9G bases, 11.9Gb downloads

Design: Libraries were constructed following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq RNA sample preparation kit (#RS-930-2001). Briefly, the poly-A containing mRNA is purified from total RNA, RNA is fragmented, double-stranded cDNA is generated from fragmented RNA, and the index containing adapters are ligated to the ends. Total RNA (2ug) was first incubated in a thermal cycler for 5 minutes at 65oC in a total volume of 50ul in a 96-well PCR plate. The plate was removed and incubated an additional 5 minutes at room temperature allowing RNA to bind to the poly-T oligo-attached magnetic beads. Beads were washed by placing the PCR plate on the magnetic stand at room temperature for 5 minutes and discarding supernatant. Bead Washing Buffer (200ul) was added and returned to the magnetic stand for 5 minutes. Supernatant was removed and discarded. The plate was removed from the magnetic stand and Elution Buffer (50ul) was added to each well. The plate was incubated at 80oC for 2 minutes and then placed at room temperature. RNA was rebound to beads with the addition of Bead Binding Buffer (50ul) and incubated for 5 minutes at room temperature. Beads were washed as previously described. First strand cDNA synthesis was performed by adding the Elute, Prime, Fragment Mix (19.5ul) to each well. The mixture was incubated for 8 minutes at 94oC. The plate was placed on the magnetic stand at room temperature for 5 minutes. From the plate, 17ul of the fragmented and primed RNA was transferred to a new PCR plate. First Strand Master Mix and Superscript II mix (8ul) was added to each well and gently mixed. Incubation was performed in a thermal cycler with the program: 25oC(10:00)+42oC(50:00)+70oC(15:00). Second strand cDNA synthesis was performed by the addition of Second Strand Master Mix (25ul) to each well. Mixture was incubated at 16oC for 1 hour. Aline PCRClean beads (90ul) were added to each well containing 50ul of ds cDNA. The plate was incubated at room temperature for 15 minutes and placed on the magnetic stand for 5 minutes. The supernatant (135ul) was removed and discarded. Each well was washed by addition of 200ul of 80% EtOH, incubation at room temperature for 30 seconds, and removal of supernatant. Wash steps were repeated once and plate was allowed to dry on magnetic stand for 15 minutes. Resuspension Buffer (52.5ul) was added to each well . The plate was returned to the magnetic stand at room temperature for 5 minutes and 50ul of supernatant was transferred to a new PCR plate. Fragment overhang ends were converted to blunt ends by the addition of the End Repair Mix (40ul) to each well and incubation at 30oC for 30 minutes. Aline PCRClean beads (160ul) were added to each well which contained 100ul of End Repair Mix. Plate was incubated at room temperature for 15 minutes. Supernatant (127.5ul) was removed and discarded. Each well was washed with 80% EtOH as previously described. The dried pellet was resuspended in Resuspension Buffer (20ul) and 15ul was transferred to a new PCR plate. The 3’ ends of the fragments were adenylated with the addition of A-Tailing Mix (12.5ul ) to each well and then incubated for 30 minutes at 37oC. DNA Ligase Mix (2.5ul) and a single RNA Adapter Mix (2.5ul) were added to each well and then incubated for 10 minutes at 37oC. The ligation reaction was stopped with the addition of Stop Ligase Mix (5ul). Aline PCRClean beads (42ul) were added to each well. The plate was incubated at room temperature for 15 minutes. Supernatant (79.5ul) was removed and discarded. Each well was washed with 80% EtOH as previously described. The dried pellet was resuspended in Resuspension Buffer (52.5ul) and 50ul was transferred to a new PCR plate. Aline PCRClean beads (50ul) were added to each well. The plate was incubated at room temperature for 15 minutes. Supernatant (95ul) was removed and discarded. Each well was washed with 80% EtOH as previously described. The dried pellet was resuspended in Resuspension Buffer (22.5ul) and 20ul was transferred to a new PCR plate. DNA fragments were enriched by adding PCR Primer Cocktail (5ul) and PCR Master Mix (25ul) to each well. PCR amplification was performed as follows: 98oC(0:30)+[98oC(0:10)+60oC(0:30)+72oC(0:30)] x 15 cycles +72oC(5:00). The amplified cDNA construct were purified by addition of Aline PCRClean beads (50ul) to each well. The plate was incubated at room temperature for 15 minutes. Supernatant (95ul) was removed and discarded. Each well was washed with 80% EtOH as previously described. The dried pellet was resuspended in Resuspension Buffer (32.5ul), incubated at room temperature for 2 minutes, and then placed on the magnetic stand for 5 minutes. Supernatant (30ul) was transferred to low binding microcentrifuge tube for storage. The final construct of each purified library was evaluated using the BioAnalyzer 2100 automated electrophoresis system, quantified with the Qubit flourometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard sequencing protocol for sequencing on the HiSeq 2000.

Submitted by: SWARTHMORE COLLEGE

Study: Trachemys scripta elegans embryonic transcriptome

PRJNA82237 • SRP011009 • All experiments • All runs

show Abstracthide Abstract

RNA was isolated from two Trachemys scripta (red-eared slider turtle) embryos (stage 14 and 17) in order to generate a T. scripta developmental transcriptome. mRNAs were reverse transcribed and the resulting cDNA pool was sequenced using an Illumina HiSeq 2000 generating 18.8Gb of sequence. Transcripts were assembled using Trinity (http://trinityrnaseq.sourceforge.net/) and annotated using Blast2GO (http://www.blast2go.com).

Sample: Trachemys scripta embryonic transcriptome

SAMN00791481 • SRS294596 • All experiments • All runs

Organism: Trachemys scripta

Library:

Name: Trachemys scripta embryonic transcriptome

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Spot descriptor:

1 forward

Runs: 1 run, 188.7M spots, 18.9G bases, 11.9Gb

Run	# of Spots	# of Bases	Size	Published
SRR412530	188,674,651	18.9G	11.9Gb	2013-01-19

ID:: 139343

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Sequence Read Archive

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