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| Status |
Public on Oct 17, 2023 |
| Title |
DNA_techrep1_mEB_bulk_v2_repB1_d22_S27 |
| Sample type |
SRA |
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| Source name |
WD44 (mouse embryonic stem cells)
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| Organism |
Mus musculus |
| Characteristics |
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 22
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| Growth protocol |
The two respective library pools (A and B, see manuscript for details) were reverse transfected (lipofectamine 2000) in mESCs. Specifically, each pool was transfected in 6 separate reactions (2M cells transfected and plated in 6 cm plates) with 400 ng hyPBase plasmid, 8 ug of reporter pool. Following recovery of cells for 2 days, cells from pairs of transfection replicates from each pool were combined (from 6 to 3 plates of cells), and the resulting three set of cells were hereafter maintained separately, constituting our biological triplicates. Puromycin (2 ug/mL) selective pressure was applied 3 days post transfection and until induction of mEBs, which happened 11 days post transfection. mEB induction proceeded as described below, with 15M of cells split in 5 plates (3M/plate) for each biological replicate. 3M of cells per replicate were also sampled for the day 0 time point. Plates from the same replicates were mixed at each medium change (once every two days). One plate’s worth of mEB was harvested on days 4, 18, 20, and 22. At harvest, cells/mEBs were fixed in 80% methanol and placed at -80C until RNA/DNA extraction for library preparation. EB induction: 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 |
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 |
genomic |
| 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.
Supplementary files format and content: bulk_MPRA_counts_mEB_v2_poolA and bulk_MPRA_counts_mEB_v2_poolB: summary count tables for bulk MPRA data. column 1: originating library short identifier, column 2: reporter architecture identifier, column 3: position of CRE relative to reporter, column 4: reporter ORF, column 5: promoter id, column 6: logical boolean indicating whether U6/oBC cassette is present on construct, column 7: logical boolean indicating whether cHS4 insulators are present on construct, column 8: pool id, column 9: CRE class, column 10: CRE id, column 11: mBC, column 12: biological replicate, column 13: technical replicate identifier, column 14: harvest day, column 15-20: 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 |
GPL30172 |
| Series (2) |
| GSE217690 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters |
| GSE245190 |
Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters [bulkMPRA (devCRE, mEB, v2)] |
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| Relations |
| BioSample |
SAMN37799320 |
| SRA |
SRX22079602 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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