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Sample GSM7791877

Query DataSets for GSM7791877

Status

Public on Mar 18, 2024

Title

Vero mock rep1

Sample type

SRA

Source name

Kidney

Organism

Chlorocebus aethiops

Characteristics

tissue: Kidney
cell line: Vero
cell type: epithelial cell
treatment: mock

Extracted molecule

other

Extraction protocol

Both infected and noninfected samples of VERO cells (ATCC CCL-81) were prepared as two biological replicates. Infected sample replicates were infected independently with MOI 5. Cell nuclei were extracted post-infection at the indicated times. Cells were washed with PBS and scraped into swelling buffer containing 10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 3 mM CaCl2, 1 U/ml SUPERase-In and 1 U/ml RNAsin plus in ultrapure water (Gibco 15564-027, Invitrogen AM9530G, Sigma-Aldrich 21115-100ml, Invitrogen 10977-035, Invitrogen AM2694, Promega N2611). Cells were centrifuged at 400 x g for 10 minutes. Formed cell pellet was resuspended into swelling buffer containing 10 % glycerol and 4 U/ml SUPERase-In (ACROS ORGANICS 327255000, Invitrogen AM2694). Nuclei were extracted by incubating the cells in swelling buffer supplemented 10 % glycerol and 1% Igepal (Igepal®CA-630 I8896-50ml) for 5 minutes. Lysis buffer composted of swelling buffer, 0.5 % Igepal, 10% glycerol, 1 U/ml SUPERase-In and 1 U/ml RNAsin plus was added to the extracted nuclei. Nuclei were washed once with lysis buffer. Isolated nuclei were resuspended into freezing buffer containing 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl (pH 8.3) and 2 U/ml SUPERase-In (Invitrogen AM9260G, Gibco 15564-027 Invitrogen 15568-025) and frozen.
Nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified using ChIP DNA Clean & Concentrator kit. The recovered cDNA was RNAseH treated, circularized, linearized, amplified for 11-14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. Libraries were sequenced for 50 cycles on an Illumina HiSeq 2000 according to the manufacturer’s instructions.

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

*library strategy: GRO-seq
Reads were trimmed using HOMER 4.3 software to remove poly-A tails and reads shorter than 25 nt were discarded. Quality control using FastQC tool, bases with poor quality were trimmed using FastX toolkit so that minimum 97 % of all bases in one read have to have a minimum phred quality score of 10. Alignments to ChlSab1.1 genome were done using bowtie-0.12.7 allowing up to two mismatches and up to three locations per read and the best alignment was reported. Differentially expressed genes were detected using ChlSab1.1.95.gtf coordinates, analyzeRNA.pl workflow of HOMER 4.3 and edgeR v3.2.2.
Assembly: ChlSab1.1
Supplementary files format and content: tab-delimited text file containing RPKMs for each sample and results from differential expression analysis

Submission date

Sep 20, 2023

Last update date

Mar 18, 2024

Contact name

Henri Niskanen

Organization name

University of Eastern Finland

Department

A.I. Virtanen Institute

Street address

P.O. Box 1627