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Sample GSM3395137 Query DataSets for GSM3395137
Status Public on Oct 12, 2018
Title SPAR-Seq of multi-exon regions of selected genes in N2A, Rbfox2 knockdown (T004), replicate 1
Sample type SRA
Source name Neuroblastoma cells
Organism Mus musculus
Characteristics cell line: N2A
cell type: Neuroblastoma
genotype/variation: Rbfox2 knockdown (T004)
Treatment protocol N2A cells were seeded in 6-well plates (500,000 cells per well) and immediately transfected with the SMARTpool siRNAs at 25 nM final concentration using RNAiMax (Invitrogen), as recommended by the manufacturer.
Growth protocol Mouse neuroblastoma (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin. All cell lines were maintained at 37°C with 5% CO2.
Extracted molecule total RNA
Extraction protocol Forty-eight hours post-transfection, total RNA were purified from cultured cells using RNeasy Mini Kit (Qiagen), as per the manufacturer’s instructions.
For each sample, a multiplex RT-PCR assay was first applied to simultaneously amplify multi-exonic regions within 108 genes in a single reaction. The multiplex RT-PCR reaction was carried out in 96-well plates using the OneStep RT-PCR kit (Qiagen) as recommended by the manufacturer, with the following changes: reactions were performed in a volume of 20 uL with 2 uL of the purified total RNA as input, and a mixture of 50 pairs of primers was added to each reaction with a final concentration of 0.25 uM for each individual forward and reverse primer. Thermocycler settings were: 50°C for 30 minutes, 95°C for 15 minute, 30 cycles of 94°C for 40 seconds, 58°C for 1 minute (slow ramp rate), 72°C for 3 minutes, and a final extension step at 72°C for 10 minutes. Two sets of barcodes were incorporated into forward and reverse primers, respectively, after the universal adaptors and added to the amplicons in the second PCR reaction to allow demultiplexing all reads that were derived from a particular treatment. Primers sequences were: forward primer, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT (N denotes i5 barcode); reverse primer, CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (N denotes i7 primer). The second PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific), as per the manufacturer’s instructions. For each 20 uL of reaction, 1 uL of the multiplex RT-PCR reaction products was used as templates. The thermal cycling conditions were as follows: 98°C for 30 seconds, 15 cycles of 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, and a final extension step at 72°C for 5 minutes. The resulting libraries were pooled and sequenced.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina MiSeq
Description Forward (i5) barcode: TAGATCGC, reverse (i7) barcode: TTGCAACG
Data processing Library strategy: SPAR-seq
Fastq files contain demultiplexed reads which were assigned to the appropriate treatment by matching to expected forward and reverse barcodes (sequenced in dedicated reads; not supplied).
Forward and reverse event reads were mapped to custom junction libraries representing all expected splice variants (supplied as processed files), using bowtie with settings --best -v 3 -k 1 --trim3 47 --trim5 5. Trimming was done to remove lower-quality, uninformative ends. Junction libraries were constructed from NCBIm37/mm9 gene annotations complemented by additional splice junctions curated from RNA-seq in the same cell type. Mapped reads were counted per junction.
Alternative splicing analysis was performed chiefly as reported in Han et al., Mol. Cell (2016): Percent-spliced-in (PSI) values were calculated for each alternative exon, or part thereof in the case of alternative 5'-/3'-splice sites ('event'), from read tallies as follows: PSI was calculated independently from forward and reverse reads as the percentage of reads supporting inclusion divided by the total number of reads for the event. For alternative 5'/3'-ss events that were part of an alternative exon (Foxm1.A2, Mta1.A1, Uspl1.A3), the PSI was instead calculated with reference to the total reads supporting inclusion of that exon in order to assess independent regulation of alternative splice site usage. Only the read direction in which all reads unambiguously supported either inclusion or exclusion of an event was used, and the average if that applied to both. Junctions used for each event are provided in the file Batch effects due to 96-well plate differences were moderated by subtracting a weighted plate median in which negative controls (siNT) were given 5x more weight than other samples. To derive variances, and because PSI values are not nearly normally distributed but roughly follow a beta distribution, a beta distribution was fit to each pair of replicate treatments, as well as to all negative controls from both replicates (siNT, untreated), using maximum-likelihood fitting as implemented in the fitdistr() function from the R package MASS {Venables & Ripley, Modern Applied Statistics with S (2002)}. Iterative optimization of shape parameters was initiated with settings x=PSI, shape1=1, shape2=1, method=”L-BFGS-B”, lower=0.01, and upper=mean number of reads supporting each PSI value. A modified Strictly Standardized Mean Difference (SSMD {Zhang, Genomics (2007)}) was then calculated based on the formula: SSMD = (μt – μc)/SQRT(vart + varc) where μt and μc are the means (corresponding to the PSI), and vart and varc the variances of the beta distributions fitted to the treatment replicates and negative controls, respectively. Events for which not all reads from at least one direction were informative were excluded from further analysis, as were events in individual treatments with less than 20 reads in one or both replicates.
Genome_build: NCBIm37/mm9
Supplementary_files_format_and_content: Processed files are either tab-delimited tables (.tab) or FASTA files (.fa). contains treatment annotations and well/plate positions. Junctions108_11-101_fwd.fa and Junctions108_11-101_rev.fa contain the custom junction upstream and downstram junction libraries, respectively, and contains the information which junctions were used to calculate PSI for each event. contains read pseudocounts (format: inclusion=exclusion) constructed from the PSI and read counts (both averged from fwd and rev junction counts, if applicable). contains raw PSI of each replicate and final SSMD.
Submission date Sep 19, 2018
Last update date Oct 12, 2018
Contact name Ulrich Braunschweig
Organization name University of Toronto
Department Donnelly Centre
Lab Benjamin J. Blencowe
Street address 160 College Street
City Toronto
State/province Ontario
ZIP/Postal code M5S 3E1
Country Canada
Platform ID GPL16417
Series (2)
GSE112601 Genome-wide CRISPR-Cas9 interrogation of splicing networks reveals a mechanism for recognition of autism-misregulated neuronal microexons
GSE120164 Genome-wide CRISPR-Cas9 interrogation of splicing networks reveals a mechanism for recognition of autism-misregulated neuronal microexons [SPAR-seq]
BioSample SAMN10089671
SRA SRX4714537

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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