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| Status |
Public on Oct 01, 2023 |
| Title |
Exosc3_mESC_cKO_isoseq_Rep2 |
| Sample type |
SRA |
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| Source name |
mESC
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| Organism |
Mus musculus |
| Characteristics |
genotype: Exosc3_cKO
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For RNA extraction, cells were washed 3x with DPBS and lysed directly on the plate with 1mL of TRIzol reagent (Invitrogen, 15596026). Subsequently, RNA was purified using Direct-zol RNA Miniprep Plus Kits (Zymo, R2072) following manufacturer instructions. In brief, lysed cells in TRIzol were mixed 1:1 with 100% ethanol. Mixed lysates were added to the Zymo-spin columns, treated with DNase I, and washed with provided buffers. RNA was eluted with UltraPure DNase/RNase-Free Distilled Water (Invitrogen, 10977015). For complementary DNA synthesis, 100-200 µg of DNase I treated RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Random hexamers were used to capture total RNA.
Purified RNA was submitted to the Icahn School of Medicine Genomic Core facility for sequencing. Sequencing libraries were prepared using SMARTer PCR cDNA Synthesis Kit (Clontech, 634926) per manufacturer recommendations. OligodT primers were used to capture full-length polyadenylated transcripts. cDNA was then size selected and sorted into two bins of greater or less than 4kb. These bins were pooled together at equimolar concentrations using SMRTbell Template Preparation Kit v1.0. End repaired and purified libraries were loaded onto a SMRTcell 1M, which was then sequenced on a Sequel I system with a 10-hour movie.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
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| Data processing |
Reads were imported into SMRTlink and IsoSeq3 software was used to obtain circular consensus sequences (CCS) for error correction, yielding highly accurate reads. Lima (v2.0.0) was subsequently used to remove barcodes, SMART-Seq primers and template-switching oligonucleotide sequences, further orienting isoforms in the correct 5’ to 3’ direction. Next, the refine command was used to remove poly(A) tails and concatamers. Reads were subsequently mapped to the mouse mm10 genome using minimap2 (v2.17) (Li, 2018) using the following parameters: -ax splice -uf -secondary=no -C5 –MD. Aligned reads were further filtered using sambamba (v0.5.7) (Tarasov et al., 2015), removing reads that are unmapped and have MAPQ < 50. Next, TranscriptClean (v.2.0.2) (Wyman and Mortazavi, 2019) as applied to correct long reads with mismatches, indels and noncanonical splice junctions not supported by spliced Illumina short reads identified from the STAR alignment described above. TALON (v5.0) (Wyman et al., 2020) was subsequently use to collapse the long reads into a novel reference transcriptome by using the talon_label_reads, talon_initialize_database, talon and talon_create_GTF commands, requiring isoforms to be at least 200bp long, and merging transcript models with matching internal splice sites but differing up to 1000bp at their 5’ or 3’ ends. Isoforms in the novel reference transcriptome were further required to have a minimum alignment threshold of 99%, a minimum sequence identity of 95%, a fraction of As ≤0.6 (removing internal priming artifacts), at least two supporting long reads, and at least one spliced short read overlapping each of the isoform’s junction in at least one short-read RNA-Seq sample. To produce a high quality reference transcriptome, isoform models classified as “incomplete splice match” or “genomic” by TALON were further discarded, as they are often products of transcript degradation or sequencing artifacts (Wyman et al., 2020).
Assembly: mm10
Supplementary files format and content: Reference transcriptome GTF; isoform counts TSV
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| Submission date |
May 31, 2022 |
| Last update date |
Oct 01, 2023 |
| Contact name |
Robert Sebra |
| E-mail(s) |
robert.sebra@mssm.edu
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| Organization name |
Icahn School of Medicine at Mount Sinai
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| Street address |
1 Gustave L. Levy Place
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL24198 |
| Series (2) |
| GSE205175 |
RNA catabolism restricts ERV expression and functionalization [Iso-seq] |
| GSE205211 |
RNA catabolism restricts ERV expression and functionalization |
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| Relations |
| BioSample |
SAMN28777539 |
| SRA |
SRX15504835 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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