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Sample GSM1714403

Query DataSets for GSM1714403

Status

Public on Sep 03, 2015

Title

Small RNA 1 hr s4U elution 2

Sample type

SRA

Source name

HEK 293T cells

Organism

Homo sapiens

Characteristics

cell line: HEK 293T
biotin reagent: MTS-biotin
s4u feed: 100 µM, 1 hr
sample type: biochemically enriched small RNA

Treatment protocol

Long RNA: Cultured cells at 80% confluence were labeled with 700 μM s4U for 60 min, followed by PBS wash, trypsinization, and harvest. Small RNA: cultured cells were split 1:8 and labeled with 100 uM s4U for 22d, 6d, 3d, 1d, 9h, 3h, 1h, 20 min, or no s4U, followed by PBS wash, trypsinization, and harvest

Growth protocol

HEK293T cells were cultured in high glucose DMEM media supplemented with 10% (v/v) fetal bovine serum, and 1% (v/v) 2 mM L-glutamine.

Extracted molecule

total RNA

Extraction protocol

Cells were resuspended in TRIzol reagent, flash frozen, and stored overnight at -80°C. Long RNA: Cell lysates were chloroform extracted once and total RNA purified by the RNeasy mini kit (Qiagen). Small RNA Cell lysates were chloroform extracted once and total RNA purified by the mirVana kit. Long and short RNA: Biotinylation and s4U-RNA enrichment with HPDP-biotin was carried out based on protocols by Gregersen et al. and adapted for MTS-biotin. Reactions were carried out in a total volume of 250 μL, containing 70 μg total RNA, 10 mM HEPES [pH 7.5], 1 mM EDTA, and 5 mg (MTSEA biotin-XX, Biotinum) or 50 mg (HPDP-biotin, Pierce) biotin dissolved in DMF (final concentration of DMF = 20%). Reactions were incubated at room temperature for 2 h (HPDP) or 30 min (MTS) in the dark. Following biotinylation, excess of free biotin was removed by phenol:chloroform extraction of the RNA using Phase-Lock-Gel tubes. RNA was precipitated at 20,000 x g for 20 min with a 1:10 volume of 5 M NaCl and an equal volume of isopropanol. The pellet was washed with an equal volume of 75% ethanol and precipitated again at 20,000 x g for 10 min. Purified RNA was dissolved in 50 uL RNase-free water and denatured at 65 °C for 10 min, followed by rapid cooling on ice for 5 min. Biotinylated RNA was separated from non-labeled RNA using μMacs Streptavidin Microbeads (Miltenyi). 200 μL beads were added to each sample and incubated for 15 min at room temperature. In the meantime, μColumns placed in the magnetic field of the μMacs separator were equilibrated with nucleic acid wash buffer supplied with the beads (Miltenyi). Reactions were applied to the μColumns and flow-through was collected as the pre-existing RNA fraction. μColumns were washed twice with 500 μL high salt wash buffer (100 mM Tris-HCl [pH 7.4], 10 mM EDTA, 1 M NaCl, and 0.1% Tween20). s4U-RNA was eluted from μColumns with 100 μL freshly prepared 100 mM DTT followed by a second elution with an additional 100 μL 5 min later. RNA was recovered from the flow-through and eluted fractions using the MinElute Spin columns (Qiagen) according to the instructions of the manufacturer. Samples were spiked with 11 ng S. pombe total RNA (a generous gift from Julien Berro) for downstream normalization.
Long RNA: 5 μg input and RNA collected from flow-through and eluted fractions were used for polyA library preparation. Sequencing libraries were constructed by standard mRNA-seq protocols by the Yale Center for Genomic Analysis (YCGA). Small RNA: 10% input from no s4U sample, along with eluted fractions were used for small RNA library preparation. Sequencing libraries were constructed by standard smRNA-seq protocols from the YCGA.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

processed data files: miRNA_quantifications_s4U.xlsx

Data processing

Sequencing reads had adapters removed using Fastx-clipper (version 0.0.13).
Reads were then mapped using Bowtie2 (version 2.2.3) separately to synthetic spikes, UniVec laboratory contaminants, and rRNAs.
Remaining reads were mapped directly to mature miRNA sequencess (miRBase 21) and then to the human genome, using sRNAbench.
Differentially expression of miRNAs between samples was evaluated using edgeR (version 3.2.4).
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files for forward and reverse strands for each sample are separated
Supplementary_files_format_and_content: Excel tables of mature miRNA quatifications and of differential expression results using edgeR package.

Submission date

Jun 17, 2015

Last update date

May 15, 2019

Contact name

Erin Elizabeth Duffy

E-mail(s)

erin_duffylacy@hms.harvard.edu

Phone

(617)432-7795

Organization name

Harvard Medical School