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Sample GSM3702736

Query DataSets for GSM3702736

Status

Public on Aug 12, 2023

Title

Lytic_LCL_input2

Sample type

SRA

Source name

EBV transformed lymphoblastoid cell line

Organism

human gammaherpesvirus 4

Characteristics

cell type: EBV transformed lymphoblastoid cell lines
passage: 12--15
rip antibody: non

Treatment protocol

To reactivate EBV, TPA (20 ng/ml) and Butyric acid (BA, 2.5 mM) were used to treat the cells for 48 hours.

Growth protocol

LCL cells were generated in our laboratory. These B-cell lines and PBMC were cultured in RPMI 1640 media (Hyclone, Logan, UT) with 10% BGS (HyClone Bovine Growth Serum).

Extracted molecule

total RNA

Extraction protocol

The total RNA extraction was performed using Trizol reagent (Invitrogen, Inc., Carlsbad, CA) and treated with DNase I (Invitrogen, Inc., Carlsbad, CA). cDNA was prepared with Superscript II reverse transcriptase kit (Invitrogen, Inc., Carlsbad, CA) according to the manufacturer’s protocol.
The library was prepared using the TruSeq stranded mRNA kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. First, the elute-frag-prime stage was done at 80°C for 2 min to allow annealing without causing fragmentation [12]. RNA was reverse transcribed into first strand cDNA using reverse transcriptase and random primers. This was followed by the second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were used for end repair process with the addition of a single ‘A’ base followed by ligation of adapters. The products were then purified and enriched by PCR amplification for 12 cycles to generate the final RNA-seq library. cDNA libraries were quantified and pooled and subsequent sequencing on an Illumina HiSeq3000 platform 50 bp single read sequencing module.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Description

LCL_lytic_vs_latent

Data processing

After sequencing, the first step is quality and adapter trimming. Trimming was conducted by Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapter sequences and low-quality bases (bases with < 20 quality score will be removed). After trimming, the MeRIP-seq reads were aligned to EBV reference genome by the aligner HISAT2 [39] with the annotation of the splice sites (the EBV reference genome and annotation were downloaded from https://www.ncbi.nlm.nih.gov/nuccore/NC_007605.1). The m6A peaks were called by a graphical model-based peak calling method – MeTPeak [40], with the parameters of window width = 50, sliding step = 50 and read length = 50. The peaks were visualized in IGV (http://www.broadinstitute.org/igv).
Genome_build: EBV genome NC_007605.1
Supplementary_files_format_and_content: peka, rpkm

Submission date

Apr 02, 2019

Last update date

Aug 12, 2023

Contact name

Fengchao Lang

E-mail(s)

flang@upenn.edu

Organization name

University of Pennsylvania

Street address

3610 Hamilton Walk