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Sample GSM6674153

Query DataSets for GSM6674153

Status

Public on Jan 05, 2023

Title

5'-Seq Replicate 4

Sample type

SRA

Source name

Yeast

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4741+ChrXVII: pCC1BAC-LCyeast-20-kb-random-DNA

Growth protocol

Yeast were grown in SD his- medium at 30˚C to OD(595)=~0.55.

Extracted molecule

polyA RNA

Extraction protocol

Hot phenol RNA extraction.
1 µg mRNA was obtained from DNased, total RNA (from the strain with random sequence artificial chromosome) via oligo d(T)25 magnetic bead enrichment. RNA was fragmented with NEBNext® Magnesium RNA Fragmentation Module and purified with ethanol precipitation. RNA was dephosphorylated with Quick CIP (NEB), heat inactivated and pre-adenylated 3’ adapter (Jin et al., 2015) was ligated with concentrated T4 RNA Ligase 1 (NEB). Reaction was split in two – mRNA deccaping enzyme (MDE, NEB) was added to one reaction, the other served as a control. 5’ adapter (Jin et al., 2015) was ligated with T4 RNA ligase 2 truncated (NEB), reaction was reverse transcribed, and the library was PCR amplified.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 2000

Description

Random DNA is at ChrXVII from 3911 to 21878 bp.

Data processing

The reads were aligned with following command: bowtie2 -x sacCer3random_spombe -5 4 -3 16 --no-mixed --no-discordant --dovetail -X 1000 (-5 4 -3 16 was used for 37 nt read length, -5 4 -3 55 was used for 76 nt long reads).
bedtools bamtobed
Bed files were used to compute fragment locations for correctly aligned paired-end reads.
Duplicates based on both read pairs and fragment location were removed from the aligned reads.
5’ isoform location was derived by trimming fragment to obtain first nt position past the 5’ adapter (or the first nucleotide of the insert between the adapters) and by computing 5’ reads per genomic coordinates. Negative control values per genomic coordinate were normalized using internal S. pombe control and subtracted from the MDE treated sample (5’ isoform samples) to obtain true 5’ isoforms.
Assembly: sacCer3 (for spike-in: S. pombe ASM294v2)
Supplementary files format and content: Replicates were analyzed individually (every MDE treated, i.e. de-capped sample has its specific control that comes from the same experiment). Normalized per nucleotide counts were combined into one file, which has 3 columns: chromosome name, position, and counts (separated into two files for two DNA strands; plus bed file aligns to the minus strand, and minus 5seq file aligns to the plus strand).
Library strategy: 5'-Seq

Submission date

Oct 24, 2022

Last update date

Jan 05, 2023

Contact name

Zlata Gvozdenov

E-mail(s)

zlata_gvozdenov@hms.harvard.edu

Organization name

Harvard Medical School

Department

BCMP