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Sample GSM1502501

Query DataSets for GSM1502501

Status

Public on Feb 27, 2015

Title

RNA-seq-HNRNPCKD-rep2

Sample type

SRA

Source name

RNA-seq-HNRNPCKD

Organism

Homo sapiens

Characteristics

cell line: HEK293T
cell type: embryonic kidney cells
genotype/variation: HNRNPC Knockdown

Growth protocol

Embryonic kidney cell line HEK293T (CRL-11268) were obtained from American Type Culture Collection (ATCC) and were cultured under standard conditions

Extracted molecule

total RNA

Extraction protocol

All PAR-CLIP RNA samples (samples 1-10) were extracted following normal PAR-CLIP procedures; the total RNA samples (samples 11-18) were extracted according to RNeasy plus kit (Catalog # 74104, Qiagen).
PAR-CLIP and PARCLIP-MeRIP libraries (Samples 1-10) were prepared using TruSeq Small RNA Sample Preparation Kit (RS-200-0012, Illumina) according to the manufacturer’s instructions, and then sequenced by Illumina Hiseq 2000 with single end 50-bp read length; RNA-seq libraries (Samples 11-18) were prepared according to the TruSeq Stranded mRNA LT Sample Prep Kit (Catalog # RS-122-9005DOC). KD and control samples were sequenced together in one flowcell on four lanes, respectively. The reads from the four lanes of each samples were combined for all analysis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample 14
processed data file: Processed_location_m6A-switch.xlsx

Data processing

The control and IP samples from PARCLIP-MeRIP experiments (same case for the control and knockdown samples from METTL knockdown experiments) were sequenced together in one flowcell on two lanes, and the reads from two lanes of each sample were combined for remaining analysis. The raw seq data was trimmed using the Trimmomatic computer program version 0.30 to remove adaptor sequences, and mapped to the Human genome version hg19 by Bowtie 1.0.0 without any gaps and allowed for at most two mismatches. For each genomic site, we calculated the average read counts within an 11-nt window centered at that site, as the normalized read counts for that site. This normalization smoothed the raw mapping curves, and facilitated identification of peaks within each mapping cluster. To correct for changes in sequencing depth or expression levels between samples, we then normalized the read counts at each genomic site to the total number of read counts on the respective gene. hnRNP C binding sites were identified by PARalyzer v1.1 with default settings.
Detection of PARCLIP-MeRIP peaks involves comparing the read counts of the IP sample with that of the control (Ctrl) sample as follows: (i) we identified all peaks within hnRNP C binding sites in the IP sample; (ii) we performed transcriptome-wide scanning to compare read counts of each identified peak in (i) with read counts at same genomic locations in the Ctrl sample to calculate the fold change score, score = log2(HIP/HCtrl). The score threshold was set to be 1, corresponding to a twofold increase compared with control. Detection of decreased hnRNP C binding sites involved comparing hnRNP C occupancies in the METTL3/14 knockdown (KD) sample with that in control as follows: (i) we identified all peaks within hnRNP C binding sites in the METTL3/14 knockdown sample; (ii) we performed transcriptome-wide scanning to compare read counts of each identified peak in (i) with read counts at the same genomic locations in control to calculate the fold change score, score = log2(HKD/HCtrl). The score threshold was set to be -1, corresponding to a twofold decrease compared with control.
RNA-seq experiments were performed on two replicate RNA samples from HNRNPC, METTL3, METTL14 KD as well as control HEK293T cells (48 hours after transfection). Total RNA samples were extracted according to RNeasy plus kit (Catalog # 74104, Qiagen). Libraries were prepared according to the TruSeq Stranded mRNA LT Sample Prep Kit (Catalog # RS-122-9005DOC). KD and control samples were sequenced together in one flowcell on four lanes, respectively. All samples were sequenced by illumina Hiseq 2000 with pair end 100-bp read length. The reads from the four lanes of each samples were combined for all analysis. The RNA-seq data was mapped using the splice-aware alignment algorithm TopHat version 1.1.4 based on the following parameters: tophat –num-threads 8 –mate-inner-dist 200 –solexa-quals –min-isoform-fraction 0 –coverage-search-segment-mismatches 1. Cufflinks was used to predict exons and cuffdiff was used to detect isoform expression level changes. Analysis of differential gene expression were performed using DESeq based on Ensembl gene annotations. Differential splicing was determined using DEXSeq based on Cufflinks-predicted, nonoverlapping exons.
Genome_build: hg19
Supplementary_files_format_and_content: Excel files with the m6A-switches sites in chromosome and transcript

Submission date

Sep 11, 2014

Last update date

May 15, 2019

Contact name

Tao Pan

E-mail(s)

taopan@uchicago.edu

Phone

(773) 702-4179

Organization name

University of Chicago