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Sample GSM4256976

Query DataSets for GSM4256976

Status

Public on Aug 11, 2020

Title

mRNA_R2

Sample type

SRA

Source name

total RNA from leaves

Organism

Solanum lycopersicum

Characteristics

tissue: Three-week-old leaves

Treatment protocol

True leaves of two weeks old plants were collected.

Growth protocol

Tomato was grown in a growth chamber under 12-h light (8:00 to 20:00, 150 μmol m-2 s-1)/12-h dark cycle at 28ºC.

Extracted molecule

total RNA

Extraction protocol

To purify the ribosome-protected fragments (RPFs) samples for cycloheximide (CHX) datasets, the polysome complexes were isolated from the grounded plant powder with polysome extraction buffer (20 mM Hepes, 100mM KCl, 5 mM MgCl2, recipe from Gao et al. (2015)) containing 100 ug/ml CHX via 13,000g at 40C for 5 min and digested with RNase 1 to purify the RPFs. The purified RPFs were further resolved in 15% TBE-UREA polyacrylamide gel (Invitrogen) and excise the gels slides corresponding to the 26~32 nt region for library construction of CHX-treated samples as described previously (Hsu et al., 2016). To purify the RNA samples for total RNA datasets, total RNAs was extracted from the aliquot of the polysome extract mentioned above via RNA clean kit (Zymo) and then input to Ribo-Zero rRNA depletion kit (Illumina) for the rRNA removal. To purify the RPFs for LTM datasets, the polysome complexes were isolated from the grounded powder of the LTM-treated plants with polysome extraction buffer via 13,000g at 40C for 5 min. The supernatant were then subjected to purimycin (PUR) treatment based a reported protocol (Gao et al., 2015) and then processed to RPF purification as described above.
The purified RNA fragments was used to construct a library for single 75-bp single-end sequencing following the protocol described previously (Ingolia, N. T., et al., Science, 2009).
Ribosome profiling (LTM-seq and CHX-seq) for initiating and elongation ribosomes ; RNA-seq for total mRNA

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Adaptor sequences (CTGTAGGCACCATCAAT) were trimmed from the sequence reads.
Genome_build: The reference genome sequences (SL3.0) and gene annotation (ITAG3.2) of Solanum Lycopersicum were retrieved from Sol Genomics Network
Supplementary_files_format_and_content: The processed data contians the number of reads of each nucleotide on the genome for 3 samples (LTM, CHX and mRNA). The "plus" and "minus"" indicated the strand direction of the seqeunced reads.

Submission date

Jan 08, 2020

Last update date

Aug 11, 2020

Contact name

Ming-Jung Liu

E-mail(s)

mjliu@gate.sinica.edu.tw

Organization name

Academia sinica

Street address

128 Sec. 2, Academia Rd, Nankang