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Sample GSM1133251

Query DataSets for GSM1133251

Status

Public on May 25, 2013

Title

LCLBACD3

Sample type

SRA

Source name

LCL cell line established using EBV B95-8 BACmid lacking ebv-mir-BHRF1-3 (D3)

Organism

Homo sapiens

Characteristics

cell line: LCL-BACD3

Growth protocol

The established lymphoblastoid cell line (LCL-BAC-D2) is infected with an EBV B95-8 BACmid (BAC) and was analyzed four months post-infection. LCL-BAC-D2 was maintained in RPMI 1640 medium supplemented with 15% FBS and 10 μg/mL gentamicin (GIBCO). LCL-BAC-D2 (mutationally inactivated for miR-BHRF1-2 expression) is infected with an EBV miRNA-mutant virus described in (Feederle, et. al., J. Vir., 2011), and were generated from the same donor as LCL-BAC, LCL-BAC-D1, and LCL-BAC-D3 (Skalsky, et. al., PloS Path., 2012; GEO GSE41437).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted with TRIzol (Life Technologies) and paired-end RNA seq libraries were generated using Ribozero and Scriptseq v2 kits (Epicentre).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

rep1

Data processing

PAR-CLIP: Libraries were stripped of the 3’ adapter sequence and 5' barcode using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads that were less than 13 nt in length or contained an ambiguous nucleotide were discarded. The small RNA files are in a tab-delimited form: SEQUENCE READCOUNT. The Ago2 PARCLIP reads were aligned to the human genome (hg19), with up to 2 mismatches allowed, by the Bowtie algorithm (Langmead et al., 2009). Mapped locations were only reported for those with the minimum number of observed mismatches for each read. All T to C mismatches between the RNA fragment and the genome were subtracted from the mismatch count for each mapped location. After subtraction only reads that mapped to a single genomic location at the minimum number of mismatches were used for further analysis. The location that a read mapped, relative to a known transcript, was determined based on the ENSEMBL v57 database 1 (Hubbard et al., 2007). If a read mapped to a location representing multiple categories, it was reported to belong to the category based on the following order of preference: 3’UTR, coding sequence, 5’UTR, intron, non-coding RNA, intergenic. Defining microRNA interaction sites by PARalyzer: Overlapping reads were grouped together for further analysis. A group must have contained ≥5 reads with conversions at two or more locations. Separate kernel density estimates were calculated across all positions based on read counts both with and without T to C conversions, as long as there was a minimum read depth of 5 at a position to ensure a robust density estimate. Clusters (*.bed files) were defined as regions for which the conversion density was greater than the non-conversion density for at least 5 consecutive nucleotides, and extended until the group read depth fell below 5. Following filtering (see supplemental methods), clusters were scored by a cross-linking index (CLI) = log2(1+T to C conversion events for all reads within a cluster). PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer
RNA-seq: RNA-seq data was aligned and quantified using RSEM with reference genome and transcript annotation [B. Li and C. Dewey (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323]. All necessary transcript annotation, genomic sequence, and alignment indices were downloaded from the Illumina iGenomes collection for Ensembl build GRCh37. Differential analysis was performed using EBseq within RSEM [Leng, N., J.A. Dawson, J.A. Thomson, V. Ruotti, A.I. Rissman, B.M.G. Smits, J.D. Haag, M.N. Gould, R.M. Stewart, and C. Kendziorski. EBSeq: An empirical Bayes hierarchical model for inference in RNA-seq experiments, UW-Madison Department of Biostatistics and Medical Informatics Paper, Bioinformatics 2012].
Genome_build: hg19
Supplementary_files_format_and_content: bed and expression

Submission date

May 03, 2013

Last update date

May 15, 2019

Contact name

Neelanjan Mukherjee

E-mail(s)

neelanjanmukherjee@gmail.com

Phone

3094061817

Organization name

MDC Berlin