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| Status |
Public on Apr 12, 2023 |
| Title |
DDR-focused pooled shRNA screen using a modified sci-RNA-seq3 [Batch1_Plate1300-2_enrich_P1-01E] |
| Sample type |
SRA |
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| Source name |
DDR-focused pooled shRNA screen using a modified sci-RNA-seq3
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| Organism |
Homo sapiens |
| Characteristics |
tissue: DDR-focused pooled shRNA screen using a modified sci-RNA-seq3
cell line: K562
cell type: lymphoblast
treatment: lentiviral transduction of a DDR-focused shRNA library
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| Treatment protocol |
Two monoclonal K562 PE2-Puro lines (clone 3 and clone 5) containing multiple randomly integrated chromatin sensors for prime editing (synHEK3) were transduced with Lenti-rtTA-P2A-Blast viruses and selected in 10 ug/mL Blasticidin for 7 days. Monoclonal lines were derived and further transduced with a DNA damage response gene-focused lentiviral shRNA library containing 304 hairpins targeting 76 genes. Cells were transdcued at either low MOI (<1) or high MOI (~10) and selected in 800 ug/mL Geneticin for 7 days. For preparing sci-RNA-seq3 libraries, at the RT step, the low MOI cells are placed in the first 6 columns of the 96-well plate, while the high MOI cells were placed in the 7-12 columns of the 96-well palte. Low MOI and High MOI transduced cells can be distiguished by RT indices.
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| Growth protocol |
K562 cells (CCL-243) were purchased from ATCC and maintained in RPMI 1640 medium (Gibco) supplied with 10% FBS (Hyclone) and penicillin/streptomycin (Gibco, 100 U/ mL).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We performed a modified version of sci-RNA-seq3 (Martin et al., 2022). In brief, K562 cells were lysed with 5 mL of a hypotonic, PBS-based lysis buffer containing DEPC. The resulting nuclei were fixed with 4 volume of ice-cold methanol. After rehydrating and washing the nuclei carefully in a sucrose/PBS/triton buffer (SPBST), the nuclei were treated with T7 polymerase in an NTP mix buffer at 37oC for 30 min. Then nuclei were washed, sonicated briefly and counted. The nuclei were distributed to a 96-well plate for reverse transcription, with a cocktail of three indexed primers: oligo-dT, HEK3 and CS1 -- specific for the cellular transcriptome, and RNAs produced from the synHEK3 reporter and the shRNA constructs, respectively. After reverse transcription, nuclei were pooled, washed in SPBST and redistributed to 4 fresh plates for ligation of the second index primer with T4 DNA ligase. Nuclei were then again pooled, washed, and redistributed to 4 final plates at two different concentrations (1300 cell per well for 2 plates and 2500 cell per well for 2 plates) for second strand synthesis and extraction. Nuclear lysates were split in half, with one half undergoing normal sci-RNA-seq3 transcriptome library preparation (tagmentation and PCR to add the third index) and the other half subjected to indexed enrichment PCR for both the synHEK3 and shRNA constructs. Products were pooled by PCR plate, size-selected and sequenced on an Illumina Nextseq 2000 or Nextseq 550.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
sci-RNA-seq3 library
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| Data processing |
For the sci-RNA-seq3 transcriptome libraries, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. Reads were filtered based on RT and ligation index, by allowing <2 hamming distance from reference. Filtered reads were trimmed with TrimGalore (v0.6.6) with parameters: -a AAAAAAAA --three_prime_clip_R1 1. Reads were then aligned to the GRCh38 reference genome using STAR (v2.7.6a). PCR duplicates were collapsed using the RT index, ligation index, UMI sequence and end coordinate of reads. Reads were further demultiplexed based on the combination of the RT, ligation and PCR index and split into files for individual cells. To generate gene expression count matrices, reads were assigned to the exonic and intronic region of closest genes with HTseq (v.2.0.2). Reads with ambiguous assignments were discarded. Cells were further filtered based on total UMI (> 100) and mitochondrial reads percentage (<10%). Cells with the number of features detected between 10% and 90% percentile of all cells were kept and considered high-quality cells. The single cell analysis was performed using the Seurat (v4.0.0) package in R.
For capture libraries of the synHEK3 and shRNA constructs, sequencing reads from the Illumina NextSeq platforms were first demultiplexed based on PCR index using the bcl2fastq software. A custom script was used to further demultiplex and filter the synHEK3 and shRNA libraries based on the combination of RT, ligation and PCR index (cell ID), by allowing <2 hamming distance from reference. For the synHEK3 library, reporter barcodes (<2 hamming distance to the reference barcode set), prime editing outcomes and read UMIs were extracted. For the shRNA library, shRNA sequences (<2 hamming distance to the reference barcode set) and read UMIs were extracted. UMIs <3 hamming distance were collapsed. A series of pre-processing steps were applied to the data. 1) We matched cell IDs between the sci-RNA-seq3 transcriptome libraries and the capture libraries, and only kept high-quality cells (number of features between 10% and 90% percentile). 2) For the shRNA library, cells with <3 or >200 UMIs or >20 shRNA were removed. 3) For the synHEK3 library, cells with <4 UMIs were first removed. We counted UMIs for synHEK3 reporters belonging to the two clones and calculated a clone UMI/total UMI ratio. If the ratio was >80% for a specific clone, the cell is assigned to the corresponding clone. Cells with extremely high UMIs (cutoff between 100 and 250 UMIs, depending on library complexity and sequencing depth of the plate) were also removed for downstream analysis. 4) For a cell-synHEK3 barcode combination, if multiple insertion sequences were observed, sequences <3 hamming distance were collapsed. Cell-synHEK3 barcode pairs having conflicting editing outcomes were discarded. 5) A cell-synHEK3 barcode-editing outcome-shRNA matrix was generated for a common set of cells identified by the shRNA and synHEK3 libraries.
Assembly: GRCh38
Supplementary files format and content: cell_synHEK3_shRNA.csv.gz contains a cell-synHEK3-shRNA capture matrix
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| Submission date |
Mar 29, 2023 |
| Last update date |
Apr 12, 2023 |
| Contact name |
Xiaoyi Li |
| E-mail(s) |
xyli10@uw.edu
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| Organization name |
University of Washington
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| Department |
Department of 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) |
| GSE228462 |
Chromatin context-dependent regulation and modulation of prime editing [sci-RNA-seq3] |
| GSE228465 |
Chromatin context-dependent regulation and epigenetic modulation of prime editing |
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| Relations |
| BioSample |
SAMN33965459 |
| SRA |
SRX19807596 |
| 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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