|
| Status |
Public on Jul 04, 2022 |
| Title |
BRCA1 BRCT PAR-Clip |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic kidney
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Embryonic kidney
cell line: HEK293T
genotype: BRCA1 BRCT
treatment: PAR-Clip by streptavidin resin and Flag resin
|
| Treatment protocol |
PAR-Clip extract BARC1/BARD1 assiciated RNA
|
| Growth protocol |
293T cells stably expressing SFB empty vector, BRCA1 BRCT or BARD1 cells were incubated with DMEM in 37℃，5% CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were incubated with 0.1 mM of 6SG for overnight followed by 0.4 Jcm2 365nm wavelength UV treatment.RNA were isolated with PAR-Clip by streptavidin resin and Flag resin.
Library preparation and sequencing were finished by Geneseed Technology CO., Ltd., Guangzhou, China. The protocol is as follows: 1) RNA quality and RNA sample assessment; 2) RNAs were fragmented, add adapters and indexes; 3) PCR; 4)Separation and cyclization; 5) Accurate quantification; 6) Sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
The gene annotation (GTF format) of CHM13 is generated with parameters: gffread chm13.draft_v1.1.gene_annotation.v4.gff3 -o chm13.draft_v1.1.gene_annotation.v4.gtf
The STAR reference of human CHM13 genome assembly v1.1 is built with parameters: STAR --runThreadN 8 --runMode genomeGenerate --genomeDir hsa_chm13v1.1_genanno_v4 --genomeFastaFiles chm13.draft_v1.1.fasta --sjdbGTFfile chm13.draft_v1.1.gene_annotation.v4.gtf --sjdbOverhang 150
The BLAST database of human ribosomal DNA is built with parameters: makeblastdb -in Hsa_rDNA_Kim.fa -dbtype nucl -out HsaKimrDNA -parse_seqids
RNA-seq reads are aligned to the pre-built human CHM13 v1.1 using STAR version 2.7.6a with the following parameters: STAR --runMode alignReads --genomeLoad NoSharedMemory --runThreadN 8 --readFilesCommand gunzip -c --genomeDir [dir_of_hsa_CHM13v1.1] --readFilesIn [read1.fq.gz] [read2.fq.gz] --chimOutJunctionFormat 1 --outReadsUnmapped Fastx --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --alignSJDBoverhangMin 10 --alignMatesGapMax 100000 --alignIntronMax 100000 --alignSJstitchMismatchNmax 5 -1 5 5 --twopassMode Basic --outFileNamePrefix [outdir_bam] --sjdbOverhang 150 --outSAMstrandField intronMotif --outSAMattributes NH HI NM MD AS XS --outSAMunmapped Within --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 50000000000 --outFilterMultimapNmax 50
Gene count data is generated using featureCounts version 2.0.2 with the following parameters: featureCounts -T 8 -p -t exon -g gene_id -a [location_of_hsa_CHM13v1.1_gtf] -o [output_featurecounts.txt] [star_align_output_sorted.bam]
The unaligned reads are converted to FASTA format and the reads shorter than 18nt are excluded using seqkit version 2.1.0: seqkit fq2fa -j 16 [unmapped_reads] | seqkit seq -m 18 -o [unmapped_reads_filtered.fa]
The unaligned sequences are aligned with human ribosomal DNA sequence (KY962518.1) using BLAST 2.11.0+ with following parameters: blastn -db [path_of_blastdb_HsaKimrDNA] -query [unmapped_reads_filtered.fa] -outfmt '6 qseqid sseqid pident length slen qlen mismatch gapopen qstart qend sstart send evalue bitscore' -out [blast_results_table]
The BLAST data are filtered, only results in 45S region are kept. These results were splitted into bins of 10nt.
Assembly: human CHM13 v1.1, and the reference sequence of human ribosomal DNA is extracted from NCBI (GenBank: KY962518.1).
Supplementary files format and content: FASTA (reference sequence of Human ribosomal DNA)
Supplementary files format and content: TXT (readpergene table generated from STAR)
Supplementary files format and content: TXT (featurecounts table generated from featurecounts)
Supplementary files format and content: TXT (10nt binned blast results)
|
| |
|
| Submission date |
Jul 01, 2022 |
| Last update date |
Jul 05, 2022 |
| Contact name |
Xiaochun Yu |
| Organization name |
Westlake University
|
| Street address |
18 Shilongshan Street
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310024 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE207338 |
BRCA1/BARD1 complex associated RNA in HEK293T cells |
|
| Relations |
| BioSample |
SAMN29471220 |
| SRA |
SRX15960884 |
