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Sample GSM2879590

Query DataSets for GSM2879590

Status

Public on Mar 01, 2019

Title

EBNA1-HA-FLAG expressing cells

Sample type

SRA

Source name

HEK293T cell line

Organism

Homo sapiens

Characteristics

cell type: Human embryonic kidney cells containing the SV40 Large T-antigen stably transfected with MSCV-EBNA1-HA-FLAG
growth protocol: Growth in DMEM/10% FBS/3ug/ml puromycin

Treatment protocol

HEK293T-EBNA1-FLAG-HA were selected in puromycin (3ug/mL)

Growth protocol

grown in DMEM (Wisent) containing 10% fetal bovine serum, cells passaged as recommended by ATCC

Extracted molecule

total RNA

Extraction protocol

HEK293T cells and HEK293T stable cells expressing EBNA1-FLAG-HA were grown upon confluency in P150 dishes, harvested and pelleted. They were then washed twice in cold PBS, and resuspended in the same volume as the pellet of polysome lysis buffer (10 mM Hepes pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 100 u/mL RNase inhibitor and complete protease inhibitor (Roche)). After an incubation of 5 minutes on ice, they were frozen at -80°C to complete the lysis. After a rapid thaw, tubes were centrifuged 20 minutes at 4°C, 13 000g. The supernatant was dosed using the Bradford assay (Thermo Scientific Coomassie Protein Assay) in triplicate. For each IP, 50 uL of Anti-Ha matrix (Roche) was centrifuged 1 min at 13,400 rpm and the supernatant was discarded. Beads were washed with 1 mL of NT2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) followed by 1 min centrifugation at 13,400 rpm for 5 times. They were then resuspended in 900 uL of NET-2 buffer (NT2 buffer supplemented with 20 mM EDTA, pH 8.0, 1 mM DTT and 100 u/mL RNase inhibitor) and 100 uL (adjusted with NT-2 buffer to a total protein quantity of 2.5 mg) of lysate from either control or EBNA1 expressing cells was added. Tubes were inverted a couple of times, centrifuged 1 min at 13,400 rpm, 4°C and 100 uL was removed as the input to evaluate RNA degradation. All tubes were incubated on a rotating wheel at 4°C overnight. Beads were precipitated at 5000g for 5 min, 4°C and washed five times with ice-cold NT2 buffer. Upon the last washing, beads were resuspended in 90 uL of NT-2 buffer and 10 uL of RQ1 DNase and incubated at 37°C for 30 minutes. Input tubes were directly added 10 uL of RQ1 DNase. Dnase-treated IP were diluted with 1 mL NT-2 buffer, centrifuged and supernatant was discarded. Beads were resuspended in 150 uL of proteinase K buffer (1% SDS, 1.2 mg/mL proteinase K from Boehringer Mannheim), and input tubes were directly added SDS and proteinase K to the same volume. Tubes were incubated at 55°C during 30 minutes with inversion at every 10 minutes. RNA was then extracted with phenol-chloroform, followed by a second chloroform extraction step. Precipitation of RNA using 272 mM ammonium acetate, 122 mM LiCl and 27 ug/mL glycogen in ice-cold ethanol was carried out during 2 hours at -80°C and followed by high-speed centrifugation. Pellets were washed in 75% ethanol, ethanol was thoroughly removed and RNA was resuspended in water.
Quality (input) and quantity (input and IP) assessments were performed on Agilent Nano Chip (Catalog number 5067-1511). Inputs from both control and EBNA1 expressing cells both showed good RNA integrity (RIN = 8.9 and 9.2, respectively), thus underlining RNA was not deteriorated due to experimental procedures. RNA was ribodepleted using Illumina Ribo-Zero rRNA Removal Kit as per manufacturer’s protocol. The RNA-seq library was then built using Illumina SSV21106 kit from 9 l ribo-depleted RNA. Library quality was assessed using Agilent DNA HS Chip (Catatalog number 5067-4626). Library quantification was performed by qPCR following Illumina Kappa library quantification protocol. HEK293T and HEK293T-EBNA1 libraries were multiplexed and sequencing was done using Illumina HiSeq 4000 at 100bp paired-end reads at McGill University and Génome Québec Innovation Centre Sequencing Service.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

RNA immunoprecipitated using anti-HA beads

Data processing

header were then remove using samtools and the custom following awk command, because they are unsuitable for the analysis with RIPSeeker: samtools view -h file.bam | awk '((NR<=197 && length($2)<10) || (NR>=198 && length($3)<5 && $3 !~ /[*]/)){print $0}' > file.out.sam The output file was then converted to the bam format using samtools and analyzed using the R package RIPSeeker. RIPSeeker, a specifically designed tool to detect enriched peaks in RIP data, was used on both HEK293T and HEK293T-EBNA1 using HEK293T as a control. Upon sequencing, 22,887,816 and 21,858,499 reads were obtained for control and EBNA1 RIP libraries, with respective average quality score of 39 and 38. Reads corresponding to both conditions were first trimmed and adaptors were removed using Trimmomatic (Galaxy Tool Version 0.32.2). STAR (version 2.5.1b) was used to align reads to hg38 human genome with annotation release 89 from the ENSEMBL database. Reads were sorted by names via samtools (version 1.3.2) and the rmdup function was used to remove PCR and sequencing duplicates. Reads unmapped or mapped to the scaffolds and their respective ID in the header were then remove using samtools (version 1.3.2) and the custom following awk command, because they are unsuitable for the analysis with RIPSeeker: samtools view -h file.bam | awk '((NR<=197 && length($2)<10) || (NR>=198 && length($3)<5 && $3 !~ /[*]/)){print $0}' > file.out.sam The output file was then converted to the bam format using samtools and analyzed using the R package RIPSeeker (version 1.10.0) . RIPSeeker, a specifically designed tool to detect enriched peaks in RIP data, was used on both HEK293T and HEK293T-EBNA1 using HEK293T as a control.
Genome_build: hg38
Supplementary_files_format_and_content: excel file from RIPSeeker containing detected peaks in EBNA1 sample using HEK293T cells as control

Submission date

Dec 07, 2017

Last update date

May 15, 2019

Contact name

Bisaillon Martin

E-mail(s)

simon.boudreault@Usherbrooke.ca

Organization name

Université de Sherbrooke

Department

Biochimie