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Status |
Public on Sep 22, 2023 |
Title |
AA_RNA_rep2 |
Sample type |
SRA |
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Source name |
cell line
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Organism |
Homo sapiens |
Characteristics |
tissue: cell line cell line: A549 genotype: WT treatment: transfected with A549 DNA
|
Treatment protocol |
The STARR-seq screening was performed as described previously (Muerdter et al. 2018). Each plasmid library was transfected into K562 or A549 cells by electroporation using Lonza SF Cell Line 4D-Nucleofector X Kit (Lonza V4XC-2012) with Lonza 4D-Nucleofector. For each library, 20 electroporation reactions were performed with 5*10^6 cells and 10μg plasmid library per reaction, following the manufactory’s protocol. After transfection, the cells were cultured for 24h at 37℃ in culture mediums without Pen/Strep. Then the cells were harvested and 3/4 of the cells were used for RNA extraction while the other 1/4 were used for plasmid DNA extraction.
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Growth protocol |
Both K562 and A549 cell lines were cultured with 1640 RPMI medium with 10% FBS, Pen/Strep (Gibco 15140-163, 500μl per 50ml) and L-Glutamine (Gibco 25030-164, 500μl per 50ml). The cells were incubated at 37℃ with 5% CO2. The K562 cells were split into 0.4*106 cells/ml when the density reached 0.8*10^6 cells/ml. The A549 cells were split 1:4 when growing into 90% confluent. Each cell type had two independent cell cultures.
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Extracted molecule |
polyA RNA |
Extraction protocol |
The RNAs were extracted using QIAGEN RNeasy Midi Kit (QIAGEN 75144) and mRNAs were selected with Invitrogen Dynabeads mRNA Purification Kit (Invitrogen 61006) following the manufactory’s protocol. The mRNAs were treated with Turbo DNase I (Ambion AM2238) and cleaned up using QIAGEN RNeasy MinElute clean up kit (QIAGEN 74204) and eluted in 60μl RNase-free water. The transfected plasmid DNA was extracted from the cells using QIAGEN Plasmid Plus Midi kit (QIAGEN 12943). Reverse transcription of the mRNAs was performed with 59μl eluted mRNA, 5μl dNTP and 1μl 10μM reporter gene specific primer: CTCATCAATGTATCTTATCATGTCTG using the Invitrogen SuperScript III First-Strand Synthesis System (Invitrogen 18080051) following the manufactory’s protocol. The cDNA was treated with RNase A and cleaned up with AMPure XP beads (Beckman A63882). Junction PCR was performed to enrich the reporter cDNA with forward primer TCGTGAGGCACTGGGCAG*G*T*G*T*C, reverse primer CTTATCATGTCTGCTCGA*A*G*C and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The sequencing ready library was generated by PCR with the junction PCR product, Universal PCR Primer for Illumina and index primer for Illumina (NEB E7335S), and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The PCR product was purified using SPRIselect beads (Beckman B23318). The sequencing ready plasmid library was generated by PCR with the extracted plasmid, Universal PCR Primer for Illumina and index primer for Illumina (NEB E7335S), and 2X Kapa HiFi HotStart ReadyMix (Kapa KK2611). The PCR product was purified using SPRIselect beads (Beckman B23318).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
AA.log2FC.bigwig AA.enhancer.bed
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Data processing |
STARR-seq data analysis: The raw sequencing data for reporter RNAs and plasmid DNAs were trimmed with Trimmomatic (version v0.39) to remove adaptors. The cleaned reads were mapped to human reference genome GRCh38 using bwa mem (version 0.7.17). The mapped reads were filtered with minimum mapping quality of 30 and minimum fragment length of 250. Potential PCR duplicates were kept. The signal tracks were generated with deeptools bamCoverage (version 3.5.2). Active enhancer analysis: To calculate enhancer activities, we split the human GRCh38 reference genome into 350bp nonoverlap bins and counted the number of RNA fragments and DNA fragments in each bin for each replicate with bedtools (version 2.31.0). We filtered the bins with DNA fragments count per million less than 1 in either replicate. Then we calculated the p-value and fold change of RNA over DNA using DESeq2 (version 1.38.3) by leveraging the two replicates of RNA and DNA and using the DNA count as control. For DESeq2, local fit type was used. Bins with raw p-value < 0.05 and log2 fold change > 0 were considered as active enhancers. Bins with p-value < 0.05 and log2 fold change < 0 were considered as negative sequences. Bins with p-value > 0.95 and the absolute value of log2 fold change < log2(1.1) were considered as neutral sequences. Assembly: GRCh38 Supplementary files format and content: bigwig: signals across the genome Supplementary files format and content: bed: peak regions
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Submission date |
Sep 22, 2023 |
Last update date |
Sep 22, 2023 |
Contact name |
Meng Wang |
E-mail(s) |
wangm@mail.cbi.pku.edu.cn
|
Organization name |
Peking University
|
Department |
School of Life Sciences
|
Street address |
No. 5 Yiheyuan Road
|
City |
Beijing |
ZIP/Postal code |
100871 |
Country |
China |
|
|
Platform ID |
GPL20301 |
Series (1) |
GSE243793 |
High-resolution dissection of human cell type-specific enhancers in cis and trans activities |
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Relations |
BioSample |
SAMN37513265 |
SRA |
SRX21858890 |