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Sample GSM1906927

Query DataSets for GSM1906927

Status

Public on Jan 11, 2016

Title

testis 2 week

Sample type

SRA

Source name

testis

Organism

Mus musculus

Characteristics

strain: FVB
age: 2 week
tissue: testis
library protocol: 3'READS

Treatment protocol

RNA were extracted from mouse testes at different developmental time points after birth, namely 1 week (w), 2w, 3w, 4w, 5w and 6w.

Growth protocol

Testis samples were obtained from mice (FVB) by surgical removal according to the approved IACUC protocols.

Extracted molecule

total RNA

Extraction protocol

The 3’ region extraction and deep sequencing (3’READS) method was previously described in [10]. Briefly, 25 μg of input RNA was used for each sample, and poly(A)+ RNA was selected using oligo d(T)25 magnetic beads (NEB), followed by on-bead fragmentation using RNase III (NEB).
Poly(A)+ RNA fragments were then selected using the chimeric U5 and T45 (CU5T45) oligo conjugated on streptavidin beads, followed by RNase H (NEB) digestion. Eluted RNA fragments were ligated with 5’ and 3’ adapters, followed by RT and PCR (15x) to obtain cDNA libraries for sequencing on the Illumina platform.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

Four random nucleotides were removed from 5'end of read. Adapter sequence were removed from the 3'end of reads by using program fastx_clipper with "-a TGGAATT". Then reads were mapped to the mouse genome using bowtie 2 (Langmead and Salzberg 2012). Reads with ≥ 2 unaligned Ts at the 5’ end are called poly(A) site-supporting (PASS) reads, which were used to identify pAs. pAs located within 24 nt from each other were clustered together. We used the Significance Analysis of Alternative Polyadenylation (SAAP) method to identify significantly regulated APA events, as previously described. Briefly, for two pAs (or two pA sets) from two comparing samples, a Relative Expression Difference (RED) score was first calculated and was called observed RED. The PASS reads were then sampled based on the assumption that the relative abundance of each pA isoform was the same in two samples. Sampling was preformed 20 times to obtain expected mean and standard deviation of RED, which were then used to convert observed RED to Z score (minus mean and divided by standard deviation). False Discovery Rate (FDR) was calculated by comparing observed Z (Zo) and expected Z (Ze) for a given Z cutoff value (Zc). For 3’UTR-APA, we selected the two most abundant pA isoforms for analysis. For CDS-APA, we combined all isoforms using pAs in upstream regions of the 3’-most exon and compared their expression change with that of isoforms using pAs in the 3’-most exon. Individual intronic pAs were also analyzed by comparing to all other pA isoforms of the gene. For uaRNA analysis, we combined all antisense transcripts using pAs within 2 kb upstream from the transcription start site (TSS), excluding those mapped to other mRNA genes, and compared them to all sense strand transcripts, excluding pAs located within 2 kb downstream of the TSS. We used FDR = 0.05 (SAAP) to select significantly regulated APA events. Global Analysis of Alternative Polyadenylation (GAAP) was previously described. Briefly, for two 3’READS data sets A and B, we sampled by bootstrapping 1.5 M PASS reads from A and B. The number of genes with significant APA changes (based on SAAP analysis) was calculated and called observed value. The data were also randomly permutated (shuffled across samples) to obtain the expected value. The observed value – expected value was normalized number of genes with significant APA regulation. This process was repeated 20 times to obtain standard deviation.
Genome_build: mm9
Supplementary_files_format_and_content: 3READS.pA.read.counts.txt contains information of gene symbol, pA coordinates, read number of pAs in different 3'READS samples. "pA_type" is pA type based on location of a pA.

Submission date

Oct 13, 2015

Last update date

May 15, 2019

Contact name

Wencheng Li

Organization name

PTC Therapeutics

Street address

100 Corporate Count

City

South Plainfield