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| Status |
Public on Oct 17, 2023 |
| Title |
RNA_techrep1_HEK293_promoters_v2_10k_rep2_S100 |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Embryonic kidney
genotype: high MOI integration via piggyBac of MPRA reporter promoter series with varying architecture. Cellular pool bottlenecked to different complexity
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| Growth protocol |
K562 cells were grown in RPMI 1640 medium (ThermoFisher, cat. num. 11875119), supplemented with 10% FBS and 1x Penicillin/streptomycin (ThermoFisher, cat. num. 15140122). HEK293T cells were grown in DMEM (ThermoFisher, cat. num. 10313021) with 10% FBS and 1x Penicillin/streptomycin. Cells were kept at 37C and 5% CO2, and passaged every two days (K562, HEK293T), except for clonal expansion. All cells were transfected in mid-exponential phase. K562 cells were transfected using Nucleofector (V4XC-2024, protocol FF12) following manufacturer’s protocol (6.5 M cells, with 9 ug reporter plasmid mix, 0.4 ug hyPBase). 2M HEK293 cells were transfected using lipofectamine 2000 (ThermoFisher) with 8 ug of reporter plasmid mix and 0.4 ug hyPBase. Cells were allowed to recover for five days post-transfection and passaged upon confluence (HEK293) or reaching 1 M/mL (K562). Following five days, 2 ug/mL puromycin selection was applied for another 7 days to select for piggyBac-mediated genomic integration of reporter transposons (which contained the GFP-P2A-puromycin resistance ORF). After dilution of the unintegrated plasmids (12 days post transfection), polyclonal cell populations were seeded at an estimated starting number of clones of 3k, 10k, and 30k each in biological duplicates. After expansion of populations to approximately 5M cells (7 to 9 days), cells were harvested, fixed in 80% methanol, and stored at -80C until extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
DNA and RNA were extracted from methanol fixed samples using the AllPrep kit (Qiagen) following manufacturer's instructions.
RNA and DNA was extracted from methanol fixed cells (AllPrep kit, Qiagen). MPRA libraries were constructed as follows: for RNA libraries, 5 ug of RNA was reverse transcribed with UMI containing primer oJBL753 using SuperScript IV (10 uL 500 ng/uL RNA mixed with 2 uL 1 uM oJBL753; 5 min at 65C, ice for >2 min; followed by addition of 4 uL 5x buffer, 1 uL 0.1M DTT, 1 uL 10 mM dNTPs, 1 uL water, 1 uL SSIV as a master mix for 20 uL total reaction; 55C for 60 min, 80C for 10 min). The cDNA was taken directly as template for PCR1 (25 uL reactions, 10 uL cDNA, primers oJBL039+Nextera v2 P7 indexed primers; 4 elongation cycles). Following the first PCR, reactions were cleaned up with 1x Ampure XP beads, eluted in 12 uL 10 mM Tris 8. 4 uL of PCR1 eluates were taken to PCR2 (10 uL reactions, primers oJBL077+oJBL359, tracked with qPCR and SYBr green, stopped at cycles 9-15 depending on reaction’s inflection point). Reactions were again purified with 1x Ampure XP and eluted in a final volume of 10 uL 10 mM Tris 8. For DNA, approximately 10 ug genomic DNA was amplified in 25 uL reactions (primers oJBL039+oJBL753; 4 cycles). Following 1x Ampure XP cleanup and elution in 12 uL 10 mM Tris 8, 4 uL of the eluate was amplified in a second round of PCR (10 uL reactions, 15 elongation cycles; primers oJBL360+Nextera v2 P7 indexed primers), cleaned up with 1x Ampure XP, and eluted in 10 uL 10 mM Tris 8. Each biological replicate was process in technical duplicate, for a total of 48 libraries (2 cell lines ⨉ 2 library types RNA/DNA ⨉ 3 bottlenecking factors ⨉ 2 biological replicates ⨉ 2 technical replicates). All reactions were performed in 96-well plates. To quantify final amplicon concentrations, a real-time qPCR experiment was performed using P5 and P7 primers (oJBL076, oJBL077 respectively). Samples were pooled according to their concentration as determined by qPCR and sequenced on a Nextseq2000 using custom set of primers (read 1: oJBL369, 15 cycles to read mBC; index 1: oJBL335 [Nextera index 1], 10 cycles to read the sample index; read 2: oJBL494 [Nextera read 2], 10 cycles to read the pseudo-UMI; index 2: oJBL371, 15 cycles to read the reverse complement of the mBC).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
Bulk MPRA
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| Data processing |
Sequencing data was demultiplexed using bcl2fastq. Raw fastq files were processed first by trimming unnecessary cycles from the 3’ end using seqtk (https://github.com/lh3/seqtk). Forward and reverse mBC reads were joined and error corrected with PEAR (options -v 15 -m 15 -n 15 -t 15). Using custom python and R scripts, successfully assembled barcode reads were combined with UMI reads, mBC/UMI pairs were counted, and the read counts and UMI count per mBC was determined.
Assembly: N/A
Supplementary files format and content: bulk_MPRA_counts_promoters_architecture_positional_effects: summary count tables for bulk MPRA data. Column 1: originating library short identifier column 2: reporter architecture identifier column 3: reporter ORF column 4: promoter id column 5: logical boolean indicating whether U6/oBC cassette is present on construct column 6: logical boolean indicating whether cHS4 insulators are present on construct column 7: mBC column 8: cell line column 9: bottleneck replicate (3k, 10k, 30k, two replicates per) column 10: technical replicate identifier column 11-16: UMI and read counts for RNA, DNA as well as normalised UMI counts (UMI divided by total UMI count in sample [individual normalization pe rsample])
Library strategy: Bulk MPRA
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| Submission date |
Oct 12, 2023 |
| Last update date |
Oct 17, 2023 |
| Contact name |
Jean-Benoit Lalanne |
| E-mail(s) |
lalannej@uw.edu
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| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Jay Shendure
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE217690 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters |
| GSE245191 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters [bulkMPRA(promoters, cells v2)] |
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| Relations |
| BioSample |
SAMN37799451 |
| SRA |
SRX22079819 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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