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| Status |
Public on Dec 11, 2022 |
| Title |
bulk_MPRA_mEB_series_mBC_repA_d08_DNA |
| Sample type |
SRA |
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| Source name |
WD44 (mouse embryonic stem cells)
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| Organism |
Mus musculus |
| Characteristics |
tissue: N/A
cell line: WD44 (mouse embryonic stem cells)
cell type: Mouse embryoid bodies
genotype: monoclonal CRISPRi (dCAS9-KRAB) cell line, high MOI (polyclonal) single-cell reporter cassette (89% devCRE library + 10% promoter series + 1% EEF1A1-mCherry) delivered by piggyBac
day: Day 08
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| Growth protocol |
Following integration of reporters via piggyBac (cells growing for >14 days for plasmid dilution), exponentially growing mESCs are lifted from the plate (aspirate medium, wash with PBS, add 2.5 mL [for 10 cm plate] 0.05% trypsin , incubate 2 minutes at 37C, deactivate trypsin and triturate to a single-cell suspension with 10 mL pre-warmed SL medium). Cells are then counted and spun down (5 min at 300 g). Supernatant is aspirated and cells are resuspended to 2 M/mL in CA medium (medium for EB induction: DMEM, 10% FBS, 1x MEM non-essential amino-acids, 1x Glutamax , 10-5 beta-mercaptoethanol). Cells are counted again, and density adjusted to 1 M/mL with CA medium. 3 mL (3 M cells) are added to 12 mL of CA medium in 10 cm plates (non gelatinized, non adherent). One the next day, plates are gently agitated to promote cell aggregation.
Following induction, embryoid bodies (mEBs) are passaged every two days (no daily medium change). mEBs are collected using a serological pipette and transferred to a 50 mL conical tube (typically three plates are pooled). Leftover mEBs on plates are recovered by a CA medium wash and pooled with in the conical tube. mEBs are left to settle (initially up to 15-20 min, faster as the mEBs grow in size). Once mEBs have settled, medium is aspirated from the top, carefully avoiding disturbing the loose pellet. Fresh, pre-warmed, CA medium is then added to 15 mL/plate and mEBs redistributed to plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following induction, mEBs were passaged every two days, with sampling 5-10% of EBs at each time point for bulk MPRA (EBs were pelleted at 5 min at 300 g, medium aspirated, fixed with ice cold 80% methanol, and stored at -80C until processing). Genomic DNA and RNA were extracted with the AllPrep kit (Qiagen). 40 samples (different replicate/batch/time points) were processed overall, comprising 13 samples repA and repB across 12 time point (day 0, 4, 6, 10, 12, 14, 16, 18, 20, 21; with two technical replicates for day 16) and 14 samples for rep2B (two technical replicates for day 0, 8, 12, 16, 18, 20; one replicate each for day 4, 6). From each sample, 2 libraries (1 gDNA-derived, 1 RNA derived) were constructed, for a total of 80 libraries. Libraries were prepared in three batches (batch1: replicates A and B, days 0, 4, 8, 12, 16; batch2: replicates A andB, days 2, 6, 10, 14, 16, 18, 20, 21; batch3: all samples from replicate 2B). For RNA, DNase treatment was applied to the first batch, but was found to be unnecessary (comparing with no reverse transcription controls), and was consequently not performed on the other two batches.
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 (one of o359-o366, o437-o448, o555-o564 per sample), 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: 1 μg of RNA was taken to reverse transcription. 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 (one of o359-o366, o437-o448, o555-o564 per sample), and reactions were stopped at inflexion point from qPCR tracking. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Each preparation batch was sequenced separately.
Batch 1: Nextseq500; read 1, mBC forward (28 cycles, primer o369); index 1, UMI (8 cycles, primer o435); read 2, mBC reverse (43 cycles, primer o371); index 2, library index (6 cycles, primer o370).
Batch 2: Nextseq2000, same set of primers (well 1: o369+o370, well2: o370+o435). read 1, mBC forward (28 cycles); index 1, UMI (10 cycles); read 2, mBC reverse (20 cycles); index 2, library index (6 cycles).
Batch 3: Nextseq500; read 1, mBC forward (18 cycles, primer o369); index 1, UMI (10 cycles, primer o435); read 2, mBC reverse (20 cycles, primer o371); index 2, library index (6 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 |
Summary count table per mBC for bulk MPRA data (time series, mouse embryoid bodies). Column 1: mBC; column 2: CRE identity; column 3: CRE class ([exogenous] promoters, devCRE); column 4: replicate ID; column 5: time point (day); column 6: preparation batch id; column 7: DNA pseudo UMI counts; column 8: RNA pseudo UMI counts; column 9: DNA read counts; column 10 RNA read counts. mBC with NA the counts were not detected in the respect experiments (=0)
Sample replicate 2B (tech rep 1) from day 20 was not included because it had clear evidence of genomic DNA signal in the RNA libraries (minimal and no promoter activity >100 higher than all other samples) likely as a result of the low RNA yield.
Supplementary files format and content: bulk_MPRA_counts_mEB_series: read and UMI counts for MPRA for each uniquely mappable barcode (including to exogenous promoters and CREs). Column 1: mBC sequence; column 2: CRE identity; column 3: CRE class (exogenous promoters: promoters, devCRE: putative CREs selected from mouse); column 4: replicate identity; column5: time point (e.g., day 6 = d06); column 6: technical library preparation batch identity; column 7: UMI counts DNA; column 8: UMI counts RNA; column 9: read counts DNA; column 10: read counts RNA.
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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 |
GPL19057 |
| Series (2) |
| GSE217679 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters [bulkMPRA_devCRE_mEBs] |
| GSE217690 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters |
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| Relations |
| BioSample |
SAMN31678045 |
| SRA |
SRX18229407 |
| 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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