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| Status |
Public on Mar 06, 2017 |
| Title |
mock_02 |
| Sample type |
SRA |
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| Source name |
GM09677 SMA fibroblasts, mock controls
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| Organism |
Homo sapiens |
| Characteristics |
treatment: mock
time point: 3days
batch: 1
cell_type: eye lens fibroblast culture
gender: male
cell line: GM09677
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| Biomaterial provider |
Coriell; http://catalog.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM09677
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| Treatment protocol |
GM09677 fibroblasts were plated a 24-well tissue culture plate at 4 x 104 cells/well in DMEM containing 10% FBS and 1x non-essential amino acids. Cells were transfected the following day at 75% confluency with Lipo2000 (Life Technologies) with either no oligo, RN-0005, or SUZ12 gapmer at a final concentration of 15 nM.
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| Growth protocol |
GM09677 fibroblasts were plated a 24-well tissue culture plate at 4 x 104 cells/well in DMEM containing 10% FBS and 1x non-essential amino acids. Cells were passaged every 2-3 days with 0.025% trypsin-EDTA to 50% confluency.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were harvested with TRIzol (Life Technologies) at either day 2 or day 3 for RNA.
RNA-Seq libraries were prepared using Illumina's TruSeq mRNA Library Prep kit according to manufacturers LT (low-throughput) protocol. Briefly, mRNA was isolated from total template RNA using oligo-dT beads, first strand cDNA was synthesized using kit provided actinomycin-D mix with added SuperScript III reverse transcriptase (Invitrogen), followed by second strand synthesis using a marking mix that incorporates dUTP in place of dTTP. 3' ends of ds-cDNA were adenylated with a single 'A' nucleotide to serve as a complementary overhang for ligation of the single 'T' of indexed Illumina sequence adapters. Adapter ligated fragments were size selected using a double-sided SPRI bead clean-up (Ampure-XP, Beckman Coulter) to target fragments in the ~300 base pair range. Purified, size selected library fragments then underwent 15 cycles of PCR amplification before a final SPRI bead clean-up to remove primer dimers. Library fragments were sized using the DNA 1000 protocol for the BioAnalyzer 2100, and final library quantification was performed using the Kapa Library Quantification qPCR Kit for Illumina (Kapa Biosystems). Libraries were then pooled, and multiplex sequencing was performed on the Illumina NextSeq 500 using a 300 cycle high output kit according to manufacturers instructions. A total of two high output runs were performed using the same multiplexed library pool in order to achieve the desired quantity of reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Fastq generation: bcl2fastq v2.17.1.14
Read quality and adapter trimming: Trimmomatic (version 0.35) modules and settings: Crop, 150 bp; IlluminaClip 2 seed mismatches, paired end seed score of 30, single end seed score of 10, minimum adapter length of 2, and keeping both reads; SlidingWindow, window size of 10 bp, minimum average phred score of 15; reads discarded if length below 36 bases.
rRNA depletion: Trimmed reads were aligned against human rRNA sequences with bowtie2 v. 2.1.0, all reads that successfully aligned were removed
Alignment: STAR v. 2.5.1a performed alignment using a modified version of hg38 reference genome that had the SMN2 containing chromosomal segment duplication (chr5:69,924,952-70,129,737) masked
Raw gene count generation: HTseq-count version 0.6.1 counted reads that overlapped features in the Gencode v24 Homo sapiens primary assembly annotation gtf (ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_24/gencode.v24.primary_assembly.annotation.gtf.gz). Parameters: mode, intersection-nonempty; minaqual, 0; stranded, reverse
Gene expression filter: Raw counts were imported into R version 3.2.3 and converted into log2 counts per million. Lowly expressed genes across the samples were filtered using a mixture model from the SCAN.UPC R package version 2.12.1
Data normalization and transformation: After filtering, the TMM normalization was applied to the data (found in the edgeR package, v 3.12.0). Count data was then transformed using the voom transform found in limma v. 3.26.7
Differential expression: Limma v 3.26.7 was used to fit the normalized, transformed data to a linear model blocking time and comparing each treatment versus the mock samples.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files for each sample include raw paired end counts for each gene (gene identifiers are based on Gencode v24 gtf file at ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_24/gencode.v24.primary_assembly.annotation.gtf.gz)
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| Submission date |
Jun 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Caroline J. Woo |
| E-mail(s) |
cwoo@ranarx.com
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| Phone |
8572423425
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| Organization name |
RaNA Therapeutics Inc.
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| Street address |
200 Sidney Street
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE83549 |
RNA-seq characterization of downstream effects of upregulating SMN2 via down-regulating PRC2 or blocking the PRC2:SMN-AS1 interaction with a mixmer oligonucleotide |
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| Relations |
| SRA |
SRX1867610 |
| BioSample |
SAMN05275975 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2209015_Sample02_GM09677_day3_mock_rep2.htseqcounts.results.txt.gz |
231.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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