title: GSM4121399: u6bsD haploid MATa TES seq YPDlog replicate 1; Saccharomyces cerevisiae; OTHER
library_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
attributes.tag: GEO Accession
attributes.value: GSM4121399