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| Status |
Public on Dec 11, 2022 |
| Title |
bulk_MPRA_promoter_series_mBC_DNA_K562_repA |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
tissue: N/A
cell line: K562
cell type: Erythroleukemia
genotype: high MOI integration via piggyBac of single-cell reporters with promoter series. Cellular pool bottlenecked to a few hundred clones
treatment: N/A
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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). HepG2 and 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) or when cells reached confluency (HepG2, typically every three days), except for clonal expansion.
All cells were transfected in mid-exponential phase. K562 cells were transfected using MaxCyte electroporation following manufacturer’s protocol (1.5 M cells, with 15 ug reporter plasmid mix (see above), 0.5 ug super piggybac transposase (SBI) in 50 uL volume). Two replicates of 1 M of HepG2 and HEK293 cells were transfected using lipofectamine 2000 (ThermoFisher) with 4 ug of reporter plasmid mix and 0.2 ug super PiggyBac transposase (SBI). Medium was changed the next day, and cells passaged as usual thereafter. After 5 days, cells were put on puromycin selection, and grown for an additional 10 days to allow complete dilution of the non integrated plasmids. At 15 days post-transfection, cells were bottlenecked to an estimated 250 and 500 starting in clones in multiple replicates, and the populations were expanded before performing bulk vs. single-cell experiments on mid-exponential cultures.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The bulk vs. single-cell quantification experiment was performed in two replicates. The first replicate (replicate A) with populations bottlenecked at an expected 250 clones, and the second replicate (replicate B) with populations bottlenecked at an expected 500 clones. For each replicate, at the same time, cells from each line were (1) harvested separately and methanol fixed for bulk quantification, and (2) prepared as single cell suspension, hand-mixed at an expected 1:1 ratio, and profiled for single-cell transcriptomics. Briefly, for the bulk methanol fixation, K562 cells (and HEK293 and HepG2 cells following lifting off plate with 0.05% trypsin) were washed once with ice cold PBS, and resuspended in 80% ice cold methanol, to a concentration of 1 M/mL, and placed at -80C until further processing.
Genomic DNA was extracted from methanol fixed cells using the DNeasy kit (Qiagen), and RNA was extracted from cells using TRIzol LS (Thermo Fisher), following manufacturer’s instructions in both cases.
Amplicon libraries from DNA were generated in two steps of PCR amplification with Kapa HiFi (Roche). 0.5-1 ug of genomic DNA input was used. For low-cycle number PCR1, gDNA was mixed with 50 μL 2⨉ Kapa HiFi master mix, 5 μL 10 μM o039, 5 μL 10 μM o358, and water to 100 μL. Cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Primer o358 contains 10 random Ns to serve as a pseudo-UMI (hereafter referred to as UMIs for brevity) to correct for PCR jackpotting. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. Illumina adapters and sequencing indices were appended through PCR2, with 4 μL of the eluate from PCR1 taken as input, and 25 μL 2⨉ Kapa HiFi master mix, 0.25 μL 100⨉ SYBr green, 2.5 μL 10 μM o077, 2.5 μL 10 μM indexed primers (o359-o364), and water to 50 μL. Libraries were amplified with tracking by qPCR with: 1 min at 95C, and cycles up to the qPCR inflection point: 20 s at 98C, 20 s at 60C, 30 s at 72C. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Amplicons libraries for RNA were obtained by first DNase treating RNA (5 μg RNA, 2 μL TURBO DNase [Thermo Fisher], 2 μL 10⨉ buffer, and water to 20 μL, incubated at 37C for 30 min, cleaned up with RNA clean & concentrator [Zymo Research], and eluted in 11 Tris 7 10 mM), and taking 1 μg of DNase treated RNA to reverse transcription. Briefly, 2 μL (500 ng/μL) RNA was mixed with 2 μL 1 μM o358, incubated at 65C for 5 min, and placed on ice. 15 μL of reverse transcription master mix was then added (4 μL 5⨉ FS buffer, 1 μL 0.1 M DTT, 1 μL 10 mM dNTP mix, 8 μL water, 1 μL SSIII [Thermo Fisher]), and the reaction incubated at 55C for 60 min, followed by 70C for 15 min. Half of the reverse transcription reaction was then directly amplified for PCR1 (37.5 2⨉ Kapa HiFi master mix, 3.75 μL o039 10 uM, 3.75 μL o077 10 uM, water to 75 μL), with cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. PCR2 proceeded as for libraries prepared from genomic DNA, with o077 and indexing primers (o365, o366, o437-o440), and reactions were stopped at inflexion point from qPCR tracking. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Read structure: read1, mBC forward (28 cycles, primer o369); index1, UMI (19 cycles, primer o435); read2, mBC reverse (19 cycles, primer o371); index2, library index (10 cycles, primer o370)
Bulk MPRA
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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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 (13 cycles from read 1, 4 cycles from read 2, 9 cycles from index 1) 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. The read and UMI counts for the mBC present in the reporter pool (determined a priori, see section on reporter cloning and subassembly) were collected for downstream analysis and comparison to single-cell quantification.
Assembly: N/A
Supplementary files format and content: bulk_MPRA_counts_promoter_series: summary count tables for bulk MPRA data (human cell lines processed separately, promoter series). Column 1: mBC; column 2: CRE identity (one of five promoters in this experiment); column 3: CRE class (all promoters for this experiment); column 4: replicate ID; column 5: cell line; column 6: DNA pseudo UMI counts; column 7: RNA pseudo UMI counts; column 8: DNA read counts; column 9 RNA read counts. mBC with NA the counts were not detected in the respect experiments (0)
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| Submission date |
Nov 10, 2022 |
| Last update date |
Dec 11, 2022 |
| 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 |
GPL18573 |
| Series (2) |
| GSE217680 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters [bulkMPRA_promoters_cell_lines] |
| GSE217690 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters |
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| Relations |
| BioSample |
SAMN31678063 |
| SRA |
SRX18229486 |
| 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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