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| Status |
Public on Aug 28, 2021 |
| Title |
VV1416_ssl2_N230D_TSS-seq_bioreplicate_2 |
| Sample type |
SRA |
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| Source name |
Saccharomyces cerevisiae cell
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: ssl2 N230D
molecule: 5' capped RNA
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| Growth protocol |
Yeast cell cultures were grown in duplicates and cells were harvested at mid-log phase at a density of 1X107 cells/mL, as determined by cell counting. For S. cerevisiae TSS-seq, cells collected from 50 mL of S. cerevisiae culture and 5 mL of S. pombe culture (serving as potential spike-in) were mixed and total RNA was extracted.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by a phenol-chloroform method originally described by Steven Hahn et.al with slight modification. Briefly, 50mL cultured yeast cells were harvested and suspended in 450uL AE (10 mM Tris-Cl; 0.5 mM EDTA, pH 9.0.) and 1% SDS solutions. Followed by immediate addition of 450uL AE-equilibrated phenol and vortex. After 65 ˚C incubation, the above mixture was transferred on sandy dry ice for 60 seconds and then moved on water ice. After spinning at 10,000rpm for 3 minutes at 4 ˚C, the aqueous layer was transferred to a tube containing 300uL AE-equilibrated phenol and 300uL of chloroform. Followed by 15 seconds of vortex and 2 minutes of spinning at 10,000, 4 ˚C. The supernatant was again transferred to another tube containing 450uL chloroform. Vertex 15 seconds and spinning 2 minutes at 10,000 rpm. The aqueous layer was carefully transferred to the third tube containing 50uL DEPC treated NaOAc and filled by 100% EtOH till the top. After 5-10 seconds of vortex, the tube was frozen at -20 ˚C for 1-2 hours. Spinning 10,000 rpm for 20 minutes at 4 ˚C. The supernatant was discarded and the pellet was washed with 75% EtOH twice and air dried. The pellet was then dissolved in 30-40uL RNase-free water and measured for concentration.
100 μg of the isolated total RNA was treated with 30 U of DNase I (QIAGEN) and purified using RNeasy Mini Kit (QIAGEN). A Ribo-Zero Gold rRNA Removal Kit (Illumina) was used to deplete rRNAs from 5 μg of DNase-treated RNAs. The rRNA-depleted RNA was purified by ethanol precipitation and resuspended in 10 μL of nuclease-free water. To remove RNA transcripts carrying a 5’ monophosphate moiety (5’-P), 2 μg of rRNA-depleted RNA were treated with 1 U Terminator 5’-Phosphate-Dependent Exonuclease (Epicentre) in the 1x Buffer A in the presence of 40 U RNaseOUT in a 50 μL reaction at 30 ˚C for 1 h. Samples were extracted with acid phenol-chloroform pH 4.5 (ThermoFisher Scientific), and RNA was recovered by ethanol precipitation and resuspended in 30 μL of nuclease-free water. Next, to remove 5’-terminal phosphates, RNA was treated with 1.5 U CIP (NEB) in 1x NEBuffer 3 in the presence of 40 U RNaseOUT in a 50 μL reaction at 37 ˚C for 30 min. Samples were extracted with acid phenol-chloroform and RNA was recovered by ethanol precipitation and resuspended in 30 μL of nuclease-free water. To convert 5’-capped RNA transcripts to 5’-monophosphate RNAs ligatable to 5’ adaptor, CIP-treated RNAs were mixed with 12.5 U CapClip (Cellscript) and 40 U RNaseOUT in 1X CapClip reaction buffer in a 40 μLreaction and incubated at 37 ˚C for 1 h. RNAs were extracted with acid phenol-chloroform, recovered by ethanol precipitation and resuspended in 10 μL of nuclease-free water. To ligate the 5’ adapter, the CapClip-treated RNA products were combined with 1 μM 5’ adapter oligonucleotide s1086 (5’-GUUCAGAGUUCUACAGUCCGACGAUCNNNNNN-3’), 1X T4 RNA ligase buffer, 40 U RNaseOUT, 1 mM ATP, 10% PEG 8000 and 10 U T4 RNA ligase 1 in a 30 μL reaction. The mixtures were incubated at 16°C for 16 h and the reactions were stopped by adding 30 μL of 2X RNA loading dye. The mixtures were separated by electrophoresis on 10% 7 M urea slab gels in 1X TBE buffer and incubated with SYBR Gold nucleic acid gel stain. RNA products migrating above the 5’ adapter oligo were recovered from the gel as described34, purified by ethanol precipitation and resuspended in 10 μL of nuclease-free water. To generate first strand cDNA, 5’-adaptor-ligated products were mixed with 0.3 μL of 100 μM s1082 oligonucleotide (5’-GCCTTGGCACCCGAGAATTCCANNNNNNNNN3’, N=A/T/G/C) containing a randomized 9-nt sequence at the 3’ end, incubated at 65 °C for 5 min, and cooled to 4 °C. A solution containing 4 μL of 5X First-Strand buffer, 1 μL (40 U) RNaseOUT, 1 μL of 10 mM dNTP mix, 1 μL of 100 mM DTT, 1 μL (200 U) of SuperScript III Reverse Transcriptase and 1.7 μL of nuclease-free water was added to the mixture. Reactions were incubated at 25 °C for 5 min, 55 °C for 60 min, 70 °C for 15 min, and cooled to 25 °C. 10 U RNase H was added, the mixtures were incubated 20 min at 37 °C and 20 μL of 2X DNA loading solution (PippinPrep Reagent Kit, Sage Science) were added. Nucleic acids were separated by electrophoresis on 2% agarose gel (PippinPrep Reagent Kit, external Marker B) to collect species of ~90 to ~550 nt. cDNA was recovered by ethanol precipitation and resuspended in 20 μL of nuclease-free water. To amplify cDNA, 9 μL of gel-isolated cDNA was added to the mixture containing 1X Phusion HF reaction buffer, 0.2 mM dNTPs, 0.25 μM Illumina RP1 primer (5’-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3’), 0.25 μM Illumina index primers RPI3-RPI16 (index primers have the same sequences on 5' and 3' ends, but different on 6-nt sequence that serves as a barcode (underlined); RPI3: 5'-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'), and 0.02 U/μL Phusion HF polymerase in 30 μL reaction. PCR was performed with an initial denaturation step of 10 s at 98 °C, amplification for 12 cycles (denaturation for 5 s at 98 °C, annealing for 15 s at 62 °C and extension for 15 s at 72°C), and a final extension for 5 min at 72 °C. Amplified cDNAs were isolated by electrophoresis on 2% agarose gel (PippinPrep Reagent Kit, external Marker B) and products of ~180 to ~550 nt were collected. cDNA was recovered by ethanol precipitation and resuspended in 13 μL of nuclease-free water. Barcoded libraries were pooled and sequenced on an Illumina NextSeq platform in high output mode using custom primer s1115 (5’-CTACACGTTCAGAGTTCTACAGTCCGACGATC-3’).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: TTS-seq
Quality control on TSS sequencing library FASTQ files was performed to remove reads with low quality using fastq_quality_filter in the FASTX-Toolkit package with parameters ‘fastq_quality_filter -v -q 20 -p 75’. Cutadapt was then used to remove the 6 nucleotide 5’ linker with parameter of ‘cutadapt -u 6’. The resulting reads were trimmed from 3’ end to 35 nucleotides long with parameter of ‘cutadapt -l 35 --minimum-length=35’. Trimmed reads were mapped to the S. cerevisiae R64-1-1 (SacCer3) genome using Bowtie with allowance of no more than two mismatches with suppression of non-uniquely mapped reads ‘bowtie -p3 -v2 -m1 -q --sam --un’, reported in sam files. Uniquely mapped reads were then extracted from sam files using SAMtools and output in bam format ‘samtools view -F 4 -S -b’. Bam files were then sorted and converted into bed files by SAMtools ‘samtools sort -o’, and BEDTools ‘bedtools bamtobed -cigar’. Customized commands were then used on bed files to identify the genomic coordinate of the 5’ end of each uniquely mapped read ‘awk 'BEGIN{FS=OFS="\t"} $6=="+" {$3=$2+1} $6=="-" {$2=$3-1} {print}'’. BEDTools was then used to determine pileup (TSS coverage) across the genome with parameters of ‘bedtools genomecov -g R64.new.genome -i -bg -strand -’ and ‘bedtools genomecov -g R64.new.genome -i -bg -strand +’, resulting in stranded bedGraph files. FASTQ files of individual library were directly processed or contacted by strains/mutants to generate begraph files for correlation analysis. For each of 5979 selected yeast promoters, TSS usage was examined within 401-nt wide window, spanning 250 nt upstream and 150 nt downstream of the previously annotated median TSS. Using customized bash and R scripts, TSS coverage from the bedGraph files of a library or a mutant were assigned into the defined windows to generate a 401×5979 TSS count table, with each row representing one of the 5979 promoters in the same order as the promoter annotation file, each column represents a promoter position, and the number in each cell representing 5’ ends mapping to that position. These count tables were stored in csv files. Using customized R script and the count table of a library or a mutant, an expression-spread-median file containing promoter expression, median TSS position of the promoter, TSS spread of the promoter was generated. The median TSS position was defined as the actual TSS containing the 50th percentile of the promoter window. The spread of TSS, which measures the width of the middle 80% of TSS distribution, was calculated by subtracting positions of 10th percentile and 90th percentile of TSS counts in 401-nt promoter window and adding 1. The positions of 10th percentile and 90th percentile of TSS counts in each promoter window were also stored in this expression-spread-median file. The streamlined codes to generate bedGraph files, the prompter annotation file and the customized scripts to generate count tables and expression-spread-median files can be found on Github account TingtingSsl2, under repository of “Ssl2_scanning”.
ChIP-exo data processing was performed as described by Rossi et al. in Nature Communications, 2018 and Qiu et al. in Genome Biology, 2020. Briefly, ChIP-exo libraries were sequenced on a NextSeq 500 in paired-end mode to generate 40 (read1) x 36 bp (read2) reads. Reads passing Q30 quality threshold were then aligned to the sacCer3 genome using the BWA-MEM alignment algorithm (v0.7.9a) with default parameters. After alignment, PCR duplicates were removed using Picard and SAMtools assuming unique combinations of read1 and read2 were PCR duplicates. Using ScriptManager v0.12, BAM files of a library were assigned into two 401×5979 matrices and saved in CDT files, which stores counts of 5’ position of protein binding on top and bottom strands, respectively. The same as in TSS-seq data analysis, each row of 401×5979 matrix representing one of 5979 promoters and each column representing a position in the 401-nt promoter window. These 401×5979 matrices were also saved in csv format. Matrices from the same mutant were combined into a single matrix by adding counts in library matrices at the same dimension and saved in csv files. Similar to TSS-seq analysis, the customized R script and the matrix of a library or a mutant were used to generate an expression-spread-median file containing promoter expression, median 5’ position of protein binding, the binding site of the spread of the promoter and saved in txt files.
Genome_build: S. cerevisiae R64-1-1 (SacCer3)
Supplementary_files_format_and_content: Each TSS-seq raw fastq file generates two begraph files. Each paired ChIP-exo fastq file generates two cdt files, one TOP strand cdt file and one BOTTOM cdt file.
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| Submission date |
Aug 25, 2021 |
| Last update date |
Aug 29, 2021 |
| Contact name |
Tingting Zhao |
| E-mail(s) |
tingtingzhao@pitt.edu
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| Phone |
9799859285
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| Organization name |
University of Pittsburgh
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| Street address |
4200 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15260 |
| Country |
USA |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE182792 |
Ssl2/TFIIH Function in Transcription Start Site Scanning by RNA Polymerase II in Saccharomyces cerevisiae |
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| Relations |
| BioSample |
SAMN16990603 |
| SRA |
SRX9629679 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5537066_VV1416_S8.csv.gz |
428.7 Kb |
(ftp)(http) |
CSV |
| GSM5537066_VV1416_S8.expression_spread_median.txt.gz |
42.6 Kb |
(ftp)(http) |
TXT |
| GSM5537066_VV1416_S8_fs.bedgraph.gz |
2.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5537066_VV1416_S8_rs.bedgraph.gz |
2.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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