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| Status |
Public on May 10, 2023 |
| Title |
heads_Isoseq_2 |
| Sample type |
SRA |
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| Source name |
heads
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: heads
developmental stage: adults
genotype: wild type (w1118)
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| Treatment protocol |
untreated
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| Growth protocol |
Flies were grown in 25C on standard apple juice based food
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| Extracted molecule |
total RNA |
| Extraction protocol |
3-day-old w1118 flies were collected and flash-frozen in liquid nitrogen and heads were used for RNA extraction.
Iso-seq libraries were prepared using 0.5µg total RNA from 3-day-old w1118 fly heads, processed with the Iso-seq express 2.0 workflow (PacBio) with 14 cycles of PCR amplification and size selection with the BluePippin system for transcripts larger than 3kb according to the manufacturer’s protocol. After SMRTbell adapter addition, libraries were sequenced on 3 SMRTcells on a Sequel I PacBio sequencer. The raw data files were processed with SMRT Link v8 software to generate CCS fastq files. Data analysis was performed using the Iso-seq3 pipeline to generate consensus reads.
Iso-seq express 2.0
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Sequel |
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| Data processing |
CIA assembly workflow: FLAIR (Tang et al., 2020) was used for transcriptome assembly. Samples were pooled to perform the assembly. For FLAIR collapse, the Eukaryotic Promoter Database EPD (Dreos et al., 2017) was provided to retain only reads with a supported TSS at their 5' end, using --max_ends 5 to allow for multiple 5’-3’ end identification. A minimum of three full-length reads were required for an isoform to be collapsed in the assembly. Assemblies were filtered for 3' ends that likely originated from internal priming or truncation during library preparation. We used FLAM-seq and RNA Direct data, as both of these methods allow for poly(A) tail detection, to perform poly(A) tail calling. Only reads containing a poly(A) tail were retained, and were trimmed to a single nucleotide preceding the poly(A) tail. Single nucleotide reads were clustered in 20-nt windows; clusters supported by at least two reads were included in the PAS database. The following filtering parameters were considered: 1) All isoforms overlapping a 3' end in a window of 100 nt were retained, 2) 3' ends found in the assembly more distal than the 3' ends found in the database were retained only if they were within the reference annotation and contained a AATAAA signal. For 3' end Correction: 1) 3’ UTR bins were created using the PAS database, starting from the end of the open reading frame, between each consecutive PAS, to the most distal PAS. Isoform 3’ ends falling within the last bin of the 3’ UTR (between the two distal-most PASs) were corrected to the most distal bin, provided the isoform covered more than 10% of the last bin.
Assembly: D. melanogaster Assembly: dm6_ensembl ; D. melanogaster Annotation: ensembl release 96
Supplementary files format and content: Processed gtf (General Transfer Format) output of FLAIR assembly: each row represents an exon. Columns are: chromosome name, source, feature type, start, end, score, strand. In the 9th column metadata assigned to each exon: gene_id, transcript_id, exon number
Library strategy: Iso-seq
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| Submission date |
May 20, 2022 |
| Last update date |
May 10, 2023 |
| Contact name |
Valérie Hilgers |
| Organization name |
MPI of Immunobiology and Epigenetics
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| Street address |
Stübeweg 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL28354 |
| Series (2) |
| GSE203497 |
Sites of transcription initiation drive mRNA isoform selection [PacBio] |
| GSE203583 |
Sites of transcription initiation drive mRNA isoform selection |
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| Relations |
| BioSample |
SAMN28572101 |
| SRA |
SRX15403485 |
| 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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