GSM4121397: Wild-type haploid MATa TES seq YPDlog replicate 1; Saccha... - SRA

SRX6996207: GSM4121397: Wild-type haploid MATa TES seq YPDlog replicate 1; Saccharomyces cerevisiae; OTHER
1 ILLUMINA (Illumina HiSeq 4000) run: 25.1M spots, 2.5G bases, 1.2Gb downloads

Submitted by: NCBI (GEO)

Study: Transcription levels of a long noncoding RNA shape a cell fate regulatory circuit (TES-seq)

PRJNA577701 • SRP225762 • All experiments • All runs

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Long noncoding RNAs (lncRNA) play diverse roles in gene regulation controlling key cellular processes, most notably, cell-fate programming {Anderson, 2016 #20;Flynn, 2014 #16;Guttman, 2011 #35;Wang, 2011 #18}. Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression { Martens, 2004 #47; Kim, 2012 #74;van Werven, 2012 #57;Hainer, 2011 #73;Kim, 2016 #41;Ard, 2016 #21;Latos, 2012 #7}. How transcription of some lncRNAs leads to activation of gene expression, while others inhibit and repress gene expression remains poorly understood {Kornienko, 2013 #17}. Here we investigated in S. cerevisiae the function of the lncRNA IRT2, which is expressed upstream in the promoter of the master regulator for entry into meiosis, IME1. We report the surprising finding that distinct levels of IRT2 transcription regulates opposing chromatin and transcription states in order to ensure that only diploids, and not haploids, enter meiosis and form gametes. In haploid cells, IRT2 transcription at very low levels is required for the correct induction of the adjacent lncRNA IRT1, which in turn represses the IME1 promoter and prevents meiotic entry {van Werven, 2012 #57}. Low levels of IRT2 transcription stimulates histone exchange delivering acetylated histone H3 lysine 56 (H3K56ac) to chromatin, thereby facilitating chromatin disassembly and recruitment of the transcriptional activator of IRT1, Rme1. Inhibiting IRT2 transcription, or mutations that resulted in cells lacking H3K56ac impairs Rme1 recruitment and IRT1 induction, and consequently haploid cells induce IME1 and undergo a lethal meiosis. In contrast to its function at low levels, increasing IRT2 transcription enhances transcription-coupled chromatin assembly and interferes with IRT1 expression, promoting IME1 expression and meiotic entry in diploid cells {Moretto, 2018 #10}. Thus, transcription of lncRNAs, even at very low levels, can play an important role in regulating gene expression, and changes in lncRNA transcription levels can confer distinct regulatory and cell fate outcomes. Overall design: 3' end RNA sequencing analysis of Saccharomyces cerevisiae SK1 strains. The SPO (low nutrient) condition correspond to strains grown in YPD to saturation, then shifted to pre-sporulation media for 16h before being transfered to sporulation media. the u6bsD mutant harbours a clean deletion (no marker) of the Ume6 binding site present 2306 bp upstream IME1 ATG. Two biological replicates of each condition were analysed. Details for library preparation are described below.

Sample: Wild-type haploid MATa TES seq YPDlog replicate 1

SAMN13032568 • SRS5517936 • All experiments • All runs

Organism: Saccharomyces cerevisiae

Library:

Instrument: Illumina HiSeq 4000

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: SINGLE

Construction protocol: Cell pellets were washed once with sterile water and snap-frozen in liquid nitrogen. Total RNA was extracted using hot acid phenol and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in RNAse-free sterile water. To obtain libraries representing the 3' ends of polyadenylated transcripts, at least 1 μg was used for 3' end sequencing. RNA fragments were mixed with 2.5 μM GsuI20TVN primer (/5BiotinTEG/ GAGCTAGTTCTGGAGTTTTTTTTTTTTTTTTTTTTVN), 0.5 mM 5-Methylcytosine-dNTPs (D1030, Zymo Research) and 0.5 μl RNasin Plus. Reaction mixtures were denatured at 65°C for 5 minutes and held at 50°C without allowing to cool. SuperScript IV, reaction buffer and 0.4 μg of Actinomycin D were added to the hot reaction mixtures and reverse transcription was performed at 50°C for 10 minutes, 80°C for 10 minutes and held at 4°C. Samples were cleaned with 1.8x volume HighPrep beads and biotinylated RNA:DNA hybrids were captured on MyOne Streptavidin C1 Dynabeads. After capture, streptavidin beads were washed once with 1x NEbuffer 2 (B7002S, NEB), re-suspended in water and subjected to second strand synthesis. The 50 μl second strand synthesis reaction consisted of 20 μl re-suspended streptavidin beads, 1X NEbuffer 2, 250 μM dNTPs, 26 μM NAD+ (B9007S), 2.5 units RNase H, 10 units E.coli DNA ligase (M0205S), and 15 units DNA polymerase I (M0209S). Second strand synthesis reactions were conducted at 16°C for 2.5 hours on a thermomixer (1400 rpm for 15 seconds, rest for 2 minutes). After reaction, beads were washed once with 1x binding and washing buffer (5.0 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1.0 M NaCl) and once with buffer B (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mg/ml BSA). Washed beads were re-suspended in 18 μl buffer B and digested with 10 units of GsuI (ER0461, ThermoFisher Scientific) at 30°C for 1 hour on a thermomixer (1400 rpm for 15 seconds, rest for 2 minutes). After digestion, the DNA fragments in the supernatant were extracted with Phenol/chloroform and precipitated at -20°C overnight in ethanol with 0.3 M sodium acetate and 1 μl linear acrylamide. Double-stranded cDNA was quantified by Qubit (Life Technologies) and used as input for library preparation using a KAPA Hyper Prep kit (KAPA Biosystems) and KAPA Single-indexed adapters (KAPA Biosystems). Gel purified libraries were quantified by Qubit and were sequenced on the Illumina HiSeq 4000 platform with 75-base single-end reads. TES-seq

Experiment attributes:

GEO Accession: GSM4121397

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 25.1M spots, 2.5G bases, 1.2Gb

Run	# of Spots	# of Bases	Size	Published
SRR10283197	25,129,635	2.5G	1.2Gb	2020-12-19

ID:: 9199217

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