title: loxPsym_mESC_Cre_CS

city: Seattle

first: Sudarshan

last: Pinglay

state: WA

address: 3720 15th Ave NE

institute: University of Washington

description: Library name: SP000237

data_processing:
10x Genomics 3’ gene expression (transcriptome) libraries were processed using cellranger-6.0.1 count function (with reference refdata-cellranger-mm10-3.0.0 for mESC cells [loxPsym+ mESC experiment 1 and attB/P+ mESC/K562 experiment lane 1] and refdata-gex-GRCh38-2020-A for K562 cells [attB/P+ mESC/K562 experiment lane 1 & 2]). Resulting raw count matrices were converted to a Seurat (v4.3.1) object using functions Read10X and CreateSeuratObject (options: min.cells=3, min.features=50). The mitochondrial fraction was computed and cell barcodes with >1500 and >1000 transcriptome UMI/cell (K562 and mESC respectively) and with 2-8% and 1.3-6% mitochondrial fraction (K562 and mESC respectively) were retained. For loxPsym+ mESC experiment 1, cell barcodes with >1000 transcriptome UMI/cell and with 1-12% mitochondrial fraction were retained. Scrublet 0.2.3 was run on filtered cells, and cells with doublet score <0.4 were retained for further analysis. For the lane 1 mESC+K562 barnyard experiment, the following criteria were further used to unambiguously assign species identity to cells: cell barcodes passing each separate species singlet processing thresholds were retained, and any cell barcodes removed as a putative scrublet-called doublet in one species but not the other were further marked as doublets (e.g., cell called as singlet in K562 mapping but flagged as a doublet in mESC mapping). Total transcriptome UMIs mapping to both species from all these cell barcodes were then inspected, showing clear separation between singlet and doublet. Initial assignment from the single species threshold was finely refined to stringently identify cells as doublets if transcriptome UMI ratios were not sufficiently separate (threshold selected by inspection): mm10/hg38 <1.42 or hg38/mm10<3.  To obtain the BC-by-cell-count matrix, we first modified the fastq files to separate the cell barcode and shuffle barcodes in two distinct files (original read structure -- read 1: first 28 cycles [cell barcode]-umi, remaining cycles 29 to 75 to read [capture sequence]-BC-[att site]; read 2: cycles 1 to 47 cycles to [read capture sequence]-BC-[att site]). Modified fastq content -- read 1: 28 cyclces [cell barcode]-umi, read 2: cycles 1 to 47 cycles to [read capture sequence]-BC-[att site] followed by original cycles 29 to 75 from original read 1. The merge was generated using seqtk version 1.4 and the following script (appending line number to join and retaining only desired information in final fastq (seqtk trimfq -b 28 file_R1_001.fastq.gz | join <(zcat file_R2_001.fastq.gz | nl) <(cat - | nl) | awk -F ' ' '{if ($2 ~ /^@VH00979/ ) {print $4" "$5;} else {print $2$3;}}' | gzip > file_w_47bpR1R2transfer_R2_001.fastq.gz).  T7-BC sequencing data was then processed using cellranger-6.0.1 count to perform error correction on the cell barcodes. The unmapped reads with error-corrected cell barcodes were selected from the sorted BAM file output, and barcodes sequences were extracted for each read from the shuffle cassette by looking for matches for constant surrounding sequences. From the read 1 portion (searching in cyles 48 to 95 appended to read 2 as described above): attP CS2 if TGAGC(.{20})GTGG, attB CS2 if TGAGC(.{20})GGCC, attP CS1 if AAAGC(.{20})TGGG, attB CS1 if AAAGC(.{20})CCGG. From the original read 2 (searching in cycles 15 to 47): attB CS1 if AAAGC(.{20})CCGG, attP CS1 if AAAGC(.{20})TGGG, attP CS2 if AAAGC(.{20})TGGG, attB CS2 if TGAGC(.{20})GGCC. The resulting two (barcode)-(capture sequence)-(recombinase site) combinations were stored and joined to the error-corrected cell barcode and umi from the read. Reads counts and total set of UMIs for all cellBC/BC1/BC2/capture/recombinase site combinations were then tallied, discarding likely chimeric UMIs (taken to be UMIs for which the proportion of reads associated to a given BC1/BC2/capture-orientation all other BC1/BC2/capture/recombinase site in the specified cell barcode falls below 0.2). The number of error-corrected UMIs for a given BC1/BC2/capture/recombinase site was then taken as the number of connected components in a graph created by connecting all UMIs associated with that combination with a Hamming distance≤1. To filter out spurious molecules (sequencing errors or PCR chimeras, only cell barcode BC1/BC2 combinations with reads/UMI ≥15 and ≥9 for Lane 1 and Lane 2 respectively were retained commensurate with the level of sequencing saturation in both libraries).
Assembly: mm10 or hg38
Supplementary files format and content: For gene expression libraries, the processed file is a seurat object. For T7 libraries, processed file is a matrix containing count of reads, UMIs per shuffle cassette barcode combination, per cell barcode.

source_name: Bl6xCast mESC

extract_protocol:
For loxPsym+ mESC scRNA-seq experiment, 300,000 parental cells were transfected in 6-well plates as described above with 1 μg of Cre and 200 ng of the reporter per transfection. 10 individual wells were transfected. At 24 hours post transfection, 5 wells were treated with 20 μM Pifithrin-α for 48 hours total. At 72 hours post transfection, both Pifithrin-α treated and untreated cells were harvested and approximately 500,000 RFP positive cells were FACS sorted into a combined tube based on the activity of the Cre reporter. Untransfected parental cells were harvested, and 1 million cells were used as input in parallel with Cre-sorted cells for the protocol.  For the attB/P+ K562 and attB/P+ mESC “Lane 1” experiment, 200,000 parental cells were nucleofected (2 reactions) or transfected (3 reactions) with 1 μg of Bxb1 plasmid as described above. All cells were harvested at day 3 post-transfection and used as input for the protocol. For the “Lane 2” experiment, attB/P+ K562 populations bottlenecked to 1000 cells after Bxb1 treatment were thawed and split once before being used as input in the protocol. For both Lanes, 500,000 cells from each population were mixed and were used for the subsequent steps.  After washing twice with cold 1X PBS (Gibco), 1 million cells per sample were resuspended in 400 μl of cold PBS. Cells were fixed with 1600 μl of cold 100% methanol, added dropwise with swirling. Cells were left on ice to fix with gentle swirling to mix every 5 minutes during the incubation. Cells were rehydrated with 4 mL of cold 1X PBS added slowly with gentle swirling of the tube. Cells were spun down and resuspended in 60 μl of PBS. For the loxPsym+ mESC experiment, approximately 60,000 cells and 380,000 cells in total were counted in the Cre and parental samples respectively using a Countess automated cell counter (Thermo) of which all and 100,000 cells in 18 μl of PBS were used for T7 IVT respectively. For the attB/P+ K562/mESC experiment, approximately 600,000 and 450,000 cells were counted of which, 100,000 cells per sample in 18 μl of PBS were used for T7 IVT. IVT reactions were set up in 30 μl total volume with the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S) with 2 μl of each NTP, buffer and enzyme. Reactions were incubated at 37°C for 1 hour in a thermocycler. Cells were immediately moved to ice and 20 μl of cold PBS was added to each sample. Approximately 40,000 cells were processed per lane of a 10X Genomics Single Cell 3’ HT with Feature Barcoding kit.
Transcriptome libraries were prepared as per the manufacturer’s protocol. To prepare libraries from T7 transcripts captured using CS1 and CS2, we started with the supernatant from the clean-up after cDNA amplification of the standard 10X Genomics feature barcoding protocol for the loxPsym+ mESC experiment. For the attB/P+ K562/mESC experiment, we spiked in two construct specific primers (oSP1059, oSP1060) at 0.5 μM into the cDNA amplification step. Two rounds of PCR were performed with primers specific to the shuffle cassette. In PCR1, oSP997 (CS1-TruSeq2), oSP1022 (CS2-TruSeq2) and oSP1061 (feature-cDNA primer F) primers were used (1.25 μl of 10 μM each) in a 50 μl Q5 polymerase reaction with 5 μl of template. Cycling conditions: 98°C - 45 s; cycles of 98°C - 20s, 60°C - 5s, 72°C - 5s; 72°C - 60s, 4°C - hold. 15 cycles of PCR1 were performed for the loxPsym+ mESC experiment and 12 cycles for the attB/P+ K562/mESC experiment. loxPsym+ mESC  were cleaned up with AmpureXP beads (1X) and eluted in 15 μl of Qiagen buffer EB. attB/P+ K562/mESC reactions were cleaned up with AmpureXP beads (1X) and eluted in 30 ul of buffer EB. In PCR2, 5 μl of PCR1 eluate was used as input into either one or two (loxPsym+ mESC experiment)  50 μl Q5 Polymerase reactions per sample with sample index primers (1.25 μl of 10 μM each) that add Illumina adapters. Reaction progress was monitored using SYBR green and was stopped before saturation. Cycling conditions for the loxPsym+ mESC experiment: 98°C - 5 min; 9 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s; 72°C - 60s, 4°C - hold. Cycling conditions for the attB/P+ K562/mESC experiment: 98°C - 1 min; 8 cycles of 98°C - 20s, 63°C - 20s, 72°C - 1min; 72°C - 60s, 4°C - hold. After a clean up with AmpureXP beads (1X), quality of the libraries was confirmed (prominent single-peak) by running them on a TapeStation D1000 ScreenTape (Agilent).  Primer sequences:  oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC oSP1061_FeaturecDNA_Primer1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG oSP1059_CS2_SpikeIn GGGGGGTTTTTTTTTTCCTTAGCC oSP1060_CS1_SpikeIn GGGGGGTTTTTTTTTTGCTAGGAC

value: Bl6xCast mESC


title: Qpct-knock out,replicate 2,snRNA-seq

status: Public on Dec 01 2024

type: SRA

city: Shanghai

first: jihong

last: zheng

state: Shanghai

address: Siping Road, Yangpu District

country: China

institute: Tongji University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Construction of fastq files were done by sequencing company
Alignment of files to reference genome using cellranger (v6.1.2) count
Assembly: mm10
Supplementary files format and content: matrix.mtx.gz contains the counts of cells called by cellranger;barcodes.tsv.gz contains the barcode name associated with each cell. The order is the same as the order of entries in matrix.mtx.gz;features.tsv.gz contains the gene name.

molecule: total RNA

organism: Mus musculus

source_name: Placenta

extract_protocol:
The nuclei of E15.5 mouse placenta (Qpct+/+ and Qpct-/-) were isolated as described in Marsh and Blelloch (2020). The placental tissue was cut into small pieces (<1mm) and placed into 2mL Nuclei EZ lysis buffer (Sigma, St. Louis, MO-NUC-101). The tissue was transferred to a dounce homogenizer with an additional 2mL Nuclei EZ lysis buffer. The nuclei were homogenized 10 times with a loose pestle (A) and 10 times with a tight pestle (B). The nuclei solution was transferred to a 15mL Falcon tube, and 2mL Nuclei EZ lysis buffer was added and incubated on ice for 5 minutes. The solution was passed through a 40μm filter and centrifuged at 500g for 8 minutes at 4℃. The supernatant was discarded, and the pellet was resuspended in 2mL Nuclei EZ lysis buffer, incubated on ice for 5 minutes, and centrifuged again at 500g for 5 minutes at 4℃. The supernatant was removed, and 2mL Nuclei Wash and Resuspension Buffer was added without mixing and incubated on ice for 5 minutes. This step was repeated once. The final nuclei solution was passed through a 35μm filter.
The isolated nuclei were loaded into a Chromium Single Cell 3’ Library & Gel Beads kit v3 i7 Multiplex kit (10× Genomics).

tag: tissue

value: Placenta

tag: cell type

value: Placenta cell

tag: age

tag: genotype

value: Qpct -/-


title: loxPsym_mESC_Parental_GEX_run2

country: USA

description: Library name: SP000233-SP000119

tag: cell line


title: loxPsym_mESC_Parental_CS

description: Library name: SP000235

library_source: other

molecule: other


title: loxPsym_mESC_Cre_GEX_run2

description: Library name: SP000234-SP000120


title: split ePE3 at multiple endogenous sites- Rep 3- I28

city: Hangzhou

first: Lijia

last: Ma

state: Zhejiang

address: Shilongshan Road No.18, Cloud Town, Xihu District

department: School of Life Sciences

institute: Westlake university

library_source: genomic

data_processing:
Traget site and protential off-target sites were amplified by PCR using the primer sequences (Table S1), then sequenced on an Illumina NovaSeq system. Deep sequencing data analysis was processed using CRISPResso2 and untreated cells were used as negative controls to exclude PCR amplification and sequencing errors. Reads were align to the amplicon sequence by crispresso2 using --max_paired_end_reads_overlap 140 --min_paired_end_reads_overlap     10 --exclude_bp_from_left 0 --exclude_bp_from_right 0 --plot_window_size 20 --min_frequency_alleles_around_cut_to_plot 0.2
On-target editing efficiencies, which are greater than 0.1%, were quantified as a percentage of (number of on-target reads) / (number of total reads).
Supplementary files format and content: xlsx files, percentage of total sequencing reads with on-target editing for all libraries, percentage of total sequencing reads with indels for ePE3-hepa.
Library strategy: Amplicon Sequencing

molecule: genomic DNA

organism: Homo sapiens

source_name: HEK293T cell

growth_protocol: HEK293T cell and Hepa1-6 cell were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108) and 1% GlutaMAX (Thermo #35050061). Cells were passaged every 2-3 days. After one day post-transduction, the medium was changed. Cells were harvested at 72 hours post-transduction.

extract_protocol:
Genomic DNA (gDNA) was isolated using TIANamp Genomic DNA Kit (TIANGEN #DP304) according to the manufacturer’s protocol.
The region that integrated with genomic DNA including RT template was PCR amplified from genomic DNA for HTS. For the first round of PCR, genomic sites of interest were amplified from 150 ng of genomic DNA using Q5 PCR Master Mix (New England Biolabs #M0544S) with the primers containing Illumina forward and reverse adapters. 50 uL of PCR 1 reaction was performed with forward and reverse primers each at 0.25 uM, ~1 uL of genomic DNA extract, and 25 uL of Q5 PCR Master Mix. To add a unique sequencing index, 50 uL of a given PCR 2 contained 2.5 uL of previous step unpurified PCR product; a unique forward or reverse primer pair, each at 0.25 uM; and 25 uL of Q5 PCR Master Mix. Primers are listed in Table S1. PCR 2 products were purified by DNA AMPure XP beads (Beckman Coulter #A63882) and eluted with 25 uL of water. DNA concentration was quantified by Nanodrop One (Thermo Fisher Scientific).

treatment_protocol: Cells were transfected at 60%-70% confluency using 3 uL of Lipofectamine 3000 (Thermo Fisher) with 1500 ng each half of the Split-PE. Three days post transfection, cells were collected.

value: HEK293T cell


title: split ePE3 at multiple endogenous sites- Rep 3- I27


title: attB/P_mESC_parental_rep2

description: Library name: SP000292

data_processing:
Reads were demultiplexed using bcl2fastq (v2.20.0.422). Read1 contains the identity of the shuffle barcodes whereas read2 contains the associated genomic sequence. Reads were passed through a custom script (IVTextractBCs.py) to extract barcode sequences based on exact matches to the expected preceding and subsequent bases. The strand of each read (top-CS2 or bottom-CS1) was also assigned at this step. PiggyBac ITR sequences were trimmed from read2 using cutadapt (v2.5) with the following parameters: -cores=4 --discard-untrimmed -e 0.2 -m 10 -a CCCTAGAAAGATA. Trimmed reads were mapped to either the mm10 or hg38 reference genome using bwa mem (v0.7.17) with -Y option. SAM files were sorted using samtools (v1.9) and filtered out reads that do not align to known PiggyBac insertions sites (TTAA) or align to several locations (contain XA:Z flag) using a custom script (align_filter.py). Filtered SAM files were converted to BED format using the sam2bed tool in bedops (v2.4.35).  For mESC samples, bedtools (v2.29.2) intersect was used with the -loj -wa -wb -filenames -sorted options to extract those alignments overlapping with a known variant between the BL6 and CAST alleles from the Sanger Mouse Genome database. A custom python script (cleanup_sort_variantcall_update.py) was used to parse the CIGAR string of each alignment intersected BED file to assign each read to each of the following categories: BL6 (read overlaps with variant and sequence of alignment at that position matches the reference allele), CAST (read overlaps with variant and sequence of alignment at that position matches variant allele) and ‘no-Variant’ (read does not overlap with variant). Inconclusive alignments that contained some incongruence in allele assignment were discarded. The position of each alignment was determined by the genomic strand it mapped to. If strand is +, position is the end of the alignment and conversely, if strand is -, position is the start of alignment. For K562 samples, alignments were simply sorted and assigned to a position using a custom python script (cleanup_sort_variantcall_update_K562.py).  Next, another custom python script (ivt_clustered_groupcollapse_iterable.py) was used to collapse alignments into groups based on: 1) a shared chromosome and position and 2) a shared set of barcodes extracted from read1. Alignments at a given position were clustered together if their associated barcodes were within a Levenshtein distance of 6. Within each cluster, the most common values for barcode1, barcode2, strand of alignment to genome (+ or -) and shuffle cassette strand (top-CS2 or bottom-CS1) were assigned as the representative values for that cluster. For mESCs, each cluster was assigned to an allele based on the most common allele value within, without taking into account the number of no-Variant assignments within that cluster. Total read count (number of alignments per cluster), total read1 UMIs (coming from forward primer of second strand synthesis), total read2 UMIs (first 8 bp of genome coming from the degenerate 3’ end of the RT primer) and total number of unique alignment lengths per cluster were also determined.
Assembly: mm10 or hg38
Supplementary files format and content: Count of reads, UMIs per shuffle cassette barcode combination clustered by sequence similarity at each chromosomal position with strand of alignment and shuffle cassette orientation. *_freq columns indicate frequency of that value in the cluster.

extract_protocol:
Qiagen DNA mini kit
The same protocol was followed both for shuffle cassette insertion site mapping from the parental population and for rearrangement call validation post Cre transfection. Genomic DNA purified using the Qiagen DNeasy kit was used as template for all IVT reactions. For loxPsym+ mESC samples, 300 ng of template, for loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 500ng of template was used per reaction. T7 IVT was performed using the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S), in a 60 μl total reaction for loxPsym+ mESCs and in a 30 μl total reaction for all other samples. Reactions were incubated at 37°C for 16 hours in a thermocycler with the lid set to 50°C. Reactions were treated with Turbo DNase (Thermo AM2238) to remove template DNA according to manufacturer’s instructions and RNA was extracted using Trizol LS reagent (Thermo 10296010). Briefly, each sample volume was normalized to 250 μl with water, 750 μl of Trizol LS reagent was added. Samples were mixed by pipetting and incubated at room temperature for 4 min. 200 μl of chloroform was added and samples were incubated at room temperature for 3 min. Samples were then spun at 12,000 x g for 15 min and the aqueous phase was transferred to a new tube. 1 μl of 5 mg/mL Glycogen was added (Invitrogen) per sample. Next, RNA was precipitated by adding 1 volume of isopropanol. Samples were mixed by inverting and incubated at -80°C for 1 hour and subsequently spun at 21,000 x g for 1 hour at 4°C. RNA pellet was washed with ice cold 80% ethanol and resuspended in 11.5 μl of H2O. Reverse transcription was performed using the SuperScript IV Reverse Transcriptase (Thermo 18090200) with 0.5 uL 100 μM RT primer that contains an 8 base-pair degenerate 3’ end (oSP1012). RNA was initially incubated with 0.5 μL 100 μM RT primer (oSP1012) and 1 μL 10 mM dNTP at 65°C for 5 min and cooled on ice. Enzyme, buffer, RNAse inhibitor and DTT were added and reactions were incubated in a thermocycler at 23°C for 10 min, 50°C for 15 min and 80°C for 10 min, followed by a hold at 10°C.  For loxPsym+ mESC replicate 1, 2nd strand synthesis for both top and bottom strands was performed in the same reaction with primers oSP1008, oSP1021 and oSP1013. For loxPsym+ mESC replicate 2, 2nd strand synthesis for top and bottom strands were performed separately, one with oSP1008 and oSP1013 and another with oSP1021 and oSP1013. Four 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with the following cycling parameters: 98°C - 3 min; 4 cycles of 98°C - 20s, 65°C - 20s, 72°C - 30s; 72°C - 60s, hold at 4°C. Reactions corresponding to a particular sample and primer pair were pooled. For replicate 1, double-sided size selection (0.5X, 1.1X) was performed with Ampure XP beads (Beckman A63882) on 200 μl of sample and eluted in 50 μl of H2O. For replicate 2, double-sided size selection (0.5X, 1.1X) was performed on 100 μl of sample and eluted in 25 μl of H2O. From each sample, a second PCR was set up with indexing primers using 12 μl of the previous eluate as input. Two 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with real-time tracking using SYBR green dye with the following cycling parameters 98°C - 3 min; 4 cycles of 98°C - 15s, 65°C - 15s, 72°C - 30s until the curves reached saturation (14-16 cycles). Running these libraries on a D1000 ScreenTape (Agilent) revealed a smear of expected size but also some lower molecular weight products that may dominate the sequencing reaction. To address this, we created 3 equimolar pools: samples from replicate 1, top strand samples from replicate 2 and bottom strand samples from replicate 2. Pools were run on a 6% TBE PAGE gel and DNA between 400 bp and 1000 bp was excised and purified.  For both replicates of loxPsym+ K562, attB/P+ mESC and attB/P+ K562 samples, 2nd strand synthesis was performed according to the replicate 2 protocol described above with minor modifications. Two 50 μl PCR 1 reactions were performed per sample, one corresponding to the top strand and one for the bottom strand. After bead clean-up, one 50 μl PCR 2 reaction was performed per sample, per strand. PCR2 was performed with the previous cycling parameters for a total of 16-18 cycles. Libraries were run on agarose gel and DNA between 400 bp and 1000 bp was excised and purified.  Library sequencing was performed on an Illumina NextSeq2000 P2 300 cycle kit. For loxPsym+ mESCs, the read lengths were: 106 read1, 10 index1, 10 index2 and 212 on read2. For all other samples, read lengths were: 124 read1, 6 index1, 10 index2 and 198 on read2.  Primer sequences:  oSP1012_IST_RT_TruSeqR2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN oSP1013_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNCCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNTTGCTAGGACCGGCCTTAAAGC


title: split ePE3 at multiple endogenous sites- Rep 3- I26


title: WT,replicate 2,snRNA-seq

description: 10x Genomics

value: Qpct +/+


title: attB/P_mESC_parental_rep1

description: Library name: SP000286


title: 200ngBxb1_d3_IVT_rep2

description: Library name: SP000036


title: WT,replicate 1,snRNA-seq

description: 10x Genomics


title: 4ugBxb1_d3_IVT_rep2

description: Library name: SP000034


title: split ePE3 at multiple endogenous sites- Rep 3- I25


title: 1ugBxb1_d3_IVT_rep2

description: Library name: SP000035


title: split ePE3 at multiple endogenous sites- Rep 3- I23


title: split ePE3 at multiple endogenous sites- Rep 3- I29


title: split ePE3 at multiple endogenous sites- Rep 3- I30


title: M_in-vivo LPS

city: Memphis

first: Michael

middle: A

last: Dyer

state: Tennessee

address: 262 Danny Thomas

department: DNB

institute: St. Jude Children's Research Hospital

data_processing:
Data was processed using Cellranger v6.0.0
Assembly: mm10-mouse / GRCh38-human
Supplementary files format and content: barcode, feautres, and matrix files for each sample

source_name: retina

extract_protocol:
Papain (40U) was added to 400uL of papain buffer and incubate at 37°C for ~10 min. Retinal punches were added to papain and incubated at 37°C for 5-10 minutes while flicking. Dnase (40uL) was added as the retina began dissociating. The suspension was transferred into a 50-ml conical tube through a 40-μm mesh cell strainer and washed with PBS−/− and layered onto a 5 ml BSA cushion medium (4% BSA in Retinal Explant media). The sample were centrifuged at 500gfor 10 min at 4 °C and the cell pellets were resuspended in REM. Cells were counted and viability was confirmed on a hemocytometer.
Libraries were constructed using 10x Genomics Chromium Single Cell 3' Reagent Kit

treatment_protocol: Retina were dissected from the eye cup in Retinal Explant Media (REM). 1.5mm punches were taken from each eye using a biopsy punchand were placed on a Nuclepore 1.0uM membrane floating on 2mL of REM in a 12-well dish. The retinal punches were incubated for 24 hours (37C, 5% CO2) for culture control retina. Punches were harvested after 24 hours.

value: retina

tag: treatment

value: 25ng LPS intravitreal injection, 24 hours retina


title: split ePE3 at multiple endogenous sites- Rep 3- I24


title: 4ugCre_d3_IVT_rep2

description: Library name: SP000031


title: 200ngCre_d3_IVT_rep2

description: Library name: SP000033


title: M_In-vivo PBS

value: 1uL PBS intravitreal injection, 24 hours retina


title: mT4 cells treated with IgG control, scRNAseq

city: Ann Arbor

first: Meredith

last: Morgan

state: Michigan

address: 1301 Catherin St

department: Radiation Oncology

institute: University of Michigan

description: 10x Genomics

data_processing:
Bcl2fastq2 Conversion Software for generating de-mulitplexed fastq files
CellRanger v7 Pipeline for read alignment and count matrices
Assembly: mm10
Supplementary files format and content: tab-seperated values files and matrix files

source_name: pancreatic tumor

growth_protocol: mT4 cells were grown in DMEM culture media with 10% fetal bovine serum. Cells were washed with PBS and trypsinized. Cells were collected and diluted to a concentration of 10 million cells/ml. 1 million cells were subcutaneously injected into 6-8 week old C57/Bl6 mice in 100ul injections.

extract_protocol:
6-8 tumors per treatment group were harvested, minced and digested in collagenase digest buffer (RPMI, Type I collagenase 0.1mg/ml, Type IV collagenase 0.1mg/ml, Dnase 2ul/ml) for 30 minutes at 37°C. Tissue digest was filtered using a 40um mesh filter and was quenched by washing with 2% FBS in PBS. Cells were counted and depleted of dying cells twice using the Dead Cell Removal kit from Miltenyi per manufacturer protocol. CD45 isolation kit from Miltenyi was used to seperate CD45- and CD45+ cells. Equal amounts of CD45- and CD45+ cells were mixed together in single cell suspension and counted once more. Samples were diluted to a final concentration of 700-1000cells/ul. Samples with greater then 85% viability were processed for sequencing. Single cell RNA-sequencing was performed at the University of Michigan Advanced Genomics Core Research Facility.
Single-cell 3' library generation was performed on the 10x Genomics Chromium Controller following the manufacturers protocol for the 3' v3.1 chemistry with NextGEM Chip G reagents (10x Genomics, Pleasanton, CA, USA). Library qualtiy was assessed by LabChip GXII HT (PerkinElmer) and wuantified by Qubit (Thermo Fisher Scientific).

treatment_protocol: Mice were treated with AZD1390 (20mg/kg) for 5 days starting on day 0, 1 dose of radiation (8Gy) on day 0, and 3 doses of anti-PD-L1 (100ug) once every three days starting on day -1. 6-8 tumors per treatment group were harvested on day 5.

value: pancreatic tumor

value: epithelial

tag: strain

value: 6-8 weeks

tag: Sex

value: female

value: Control


title: Qpct-knock out,replicate 1,snRNA-seq

description: 10x Genomics


title: M_WT Retina_3

value: WT Retina


title: split ePE3 at multiple endogenous sites- Rep 3- I22


title: parental_IVT_rep2

description: Library name: SP000030


title: Flu_otv_pig_ln2, replicate 1, scRNA-seq

city: Columbia

first: John

last: Driver

state: MO

address: 1201 Rollins Street

institute: University of Missouri

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, and gene counting were made using the Cell Ranger software v7.0.1
Assembly: Sscrofa11.1
Supplementary files format and content: Tab-separated values files and matrix files

organism: Sus scrofa

source_name: Lymph node

extract_protocol:
Approximately 1 g of tissue collected from the left cranial, middle, and caudal lung lobes were combined and digested with 2.5 mg/mL of Liberase TL in Dulbecco’s Modified Eagle Medium at 37oC for 45 minutes, passed through a 100 micrometer cell strainer, and treated with an ammonium chloride-based red blood cell lysis buffer. The resulting cell suspensions were washed, stained with propidium iodide for exclusion of dead cells, and FACS sorted for live cells using a Sony SH800 Cell Sorter.
Gene expression library was performed according to the manufacturer's instructions (single cell 3' protocol, 10x Genomics)

treatment_protocol: Pigs were intratracheally infected with 10^6 TCID50 pH1N1 and treated, or mock treated, with 75mg of oseltamivir phosphate BID during 4 days.

value: Lymph node

value: leukocytes

value: Influenza-infected, oseltamivir-treated


title: Flu_otv_pig_ln1, replicate 1, scRNA-seq

description: 10x Genomics


title: M_WT Retina_2


title: split ePE3 at multiple endogenous sites- Rep 3- I21


title: 200ngBxb1_d3_IVT_rep1

description: Library name: SP000029

value: Bl6xCast mESC


title: M_WT Retina_1


title: Flu_pig_ln3, replicate 1, scRNA-seq

description: 10x Genomics

value: Influenza-infected, mock-treated


title: split ePE3 at multiple endogenous sites- Rep 3- I20


title: 1ugBxb1_d3_IVT_rep1

description: Library name: SP000028


title: 1ugCre_d3_IVT_rep2

description: Library name: SP000032


title: Flu_otv_pig_ln3, replicate 1, scRNA-seq

description: 10x Genomics


title: Flu_pig_ln2, replicate 1, scRNA-seq

description: 10x Genomics


title: 4ugBxb1_d3_IVT_rep1

description: Library name: SP000027


title: M_Low Oxygen_2

value: Retina, 24 hour culture in REM at 2.3% Oxygen


title: M_MeOH_2

value: Retina, 24 hour culture in REM + 8uM Formic Acid


title: 200ngCre_d3_IVT_rep1

description: Library name: SP000026


title: Flu_otv_pig_lung5, replicate 1, scRNA-seq

description: 10x Genomics

source_name: Lung

value: Lung


title: split ePE3 at multiple endogenous sites- Rep 3- I17

value: HEK293T cell


title: M_Control_8

value: retina

value: Retina, 24 hour culture in REM


title: Flu_pig_ln1, replicate 1, scRNA-seq

description: 10x Genomics


title: 1ugCre_d3_IVT_IVT_rep1

description: Library name: SP000025


title: Flu_otv_pig_lung4, replicate 1, scRNA-seq

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 3- I16


title: 4ugCre_d3_IVT_rep1

description: Library name: SP000024


title: split ePE3 at multiple endogenous sites- Rep 3- I15


title: M_C. Albicans

value: Retina, 24 hour culture in REM + 100CFU/mL C. Albicans


title: Flu_otv_pig_lung2, replicate 1, scRNA-seq

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 3- I14


title: split ePE3 at multiple endogenous sites- Rep 3- I19


title: M_Control_7


title: split ePE3 at multiple endogenous sites- Rep 3- I13


title: Flu_otv_pig_lung1, replicate 1, scRNA-seq

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 3- I18


title: M_pH 7.15

value: Retina, 24 hour culture in REM at pH 7.15


title: M_pH 7.69

value: Retina, 24 hour culture in REM at pH 7.69


title: M_IFN_2

value: Retina, 24 hour culture in REM + 100U/mL IFN g


title: Flu_pig_lung5, replicate 1, scRNA-seq

description: 10x Genomics


title: Flu_otv_pig_lung3, replicate 1, scRNA-seq

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 3- I12


title: parental_IVT_rep1

description: Library name: SP000023


title: Flu_pig_lung4, replicate 1, scRNA-seq

description: 10x Genomics


title: attB/P_mESC_parental_rep2

description: Library name: SP000319

data_processing:
Reads were demultiplexed using bcl2fastq (v2.20.0.422). Reads were passed through a custom script (read_extract_iterable.py or read_extract_iterable_4primers.py or amplicon_BCExtract_20240906.py) to extract barcode sequences and UMIs based on matches to the expected preceding and subsequent bases. In the case of 4-primer amplicon-seq, the strand of each read (top-CS2 or bottom-CS1) was also assigned at this step. The identity of the recombination site (loxP, attB, attP, attL or attR) was determined from the combination of bases specific to each site expected to be detected in read 1 and read 2.Total number of reads per barcode pair was taken as the readcount and the total number of unique UMIs detected was taken as the UMI count (df_group.py or df_group_4primers.py). In 4-primer amplicon-seq, the same shuffle cassette can result in two distinct amplicons. We collapsed reads coming from the same shuffle cassette based on the shared set of barcodes and summed up the read counts and UMI counts. Those barcode pairs where we did not detect both types of amplicons were discarded. In all cases, the barcode closest to CS2 was named barcode1 and the barcode closest to CS1 in the shuffle cassette was named barcode2.
Supplementary files format and content: Count of reads, UMIs per shuffle cassette barcode combination without normalization for sequencing depth.

extract_protocol:
Qiagen DNA mini kit
We employed two different strategies for amplicon-seq library construction: 2-primer  and 4-primer. The major difference between these two strategies is that either 2 or 4 primers corresponding to the capture sequences were used to generate the PCR product. In the case of the 2-primer strategy, the P5 Illumina sequencing adapter can only come from the primer that binds CS2 and the P7 adapter can only come from the primer that binds CS1. This precludes identification of recombined shuffle cassettes that contain the same capture sequence on both sides of the loxPsym site. In the 4 primer strategy, primers with P5 and P7 adapters that bind to both CS2 and CS1 are included in the PCR. Theoretically, this would enable us to detect recombined shuffle cassettes with the same capture-sequence on both sides. Two rounds of PCR were performed on genomic DNA extracted from indicated samples to construct sequencing libraries. For the 2-primer experiments, we used 250 ng of genomic DNA prepared using the Qiagen DNeasy kit as input. PCR1 (UMI addition) was performed in a 50 μl reaction with Q5 Polymerase (NEB M0492S), with 2.5 μL of each 10 μM primer (oSP1008 and oSP997) with the following cycling parameters: 98°C - 5 min; 4 cycles of 98°C - 20s, 62°C - 20s, 72°C - 30s; 72°C - 60s. PCRs were cleaned up using AmpureXP beads (1X) and eluted in 11 μl of H2O. PCR2 (sample indexing and sequencing adapter addition) was again performed with Q5 Polymerase in a 50 μl reaction using 10 μl of eluate from the previous step as template and 2.5 μL of each 10 μM sample indexing primer. Progression of PCR2 was monitored in real-time using SYBR green dye and reactions were stopped before saturation (usually 15-18 cycles). Cycling conditions for PCR2:  98°C - 5 min; 15 cycles of 98°C - 10s, 65°C - 10s, 72°C - 20s. Reactions were cleaned up with AmpureXP beads (1X) and eluted in 12 μl of H2O. Sample quality was confirmed on a TapeStation D1000 ScreenTape (Agilent), after which reactions were pooled. Sequencing was performed on an Illumina NextSeq2000 100 cycle kit with the following read lengths: 69 read1, 6 index1, 10 index2 and 53 on read2. For the 4-primer experiments involving loxPsym+ mESCs, 100 ng of genomic DNA prepared using the Qiagen DNeasy kit as used as input. For the 4-primer experiments involving loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 250ng of genomic DNA prepared using the Qiagen DNeasy kit was used as input. PCR1 was performed with a mix of 4 primers (oSP1008, oSP997, oSP1021 and oSP1022) with 0.125 μl of each primer at 100 μM per 50 μl reaction. The rest of the protocol was identical to the 2-primer workflow described above. Primer sequences: oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN CCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNN TTGCTAGGACCGGCCTTAAAGC oSP997_CS1_TruSeq2_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC TTGCTAGGACCGGCCTTAAAGC oSP1022_CS2_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTTAGCCGCTAATAGGTGAGC


title: attB/P_mESC_Bxb1_d3_rep2

description: Library name: SP000320


title: split ePE3 at multiple endogenous sites- Rep 3- I11


title: Flu_pig_lung3, replicate 1, scRNA-seq

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 3- I10


title: M_Glutamate

value: Retina, 24 hour culture in REM + 1mM Glutamate


title: attB/P_mESC_Bxb1_d7_rep1

description: Library name: SP000261


title: Flu_pig_lung2, replicate 1, scRNA-seq

description: 10x Genomics


title: attB/P_mESC_Bxb1_d7_rep2

description: Library name: SP000322


title: split ePE3 at multiple endogenous sites- Rep 3- I9


title: attB/P_mESC_Bxb1_d5_rep1

description: Library name: SP000258


title: M_Ouabain

value: Retina, 24 hour culture in REM + 70uM Ouabain


title: Healthy_pig_lung2, replicate 1, scRNA-seq

description: 10x Genomics

value: Mock-infected


title: Flu_pig_lung1, replicate 1, scRNA-seq

description: 10x Genomics

value: leukocytes


title: split ePE3 at multiple endogenous sites- Rep 3- I8


title: split ePE3 at multiple endogenous sites- Rep 3- I7


title: attB/P_mESC_Cre_d5_rep1

description: Library name: SP000257


title: attB/P_mESC_Bxb1_d3_rep1

description: Library name: SP000254


title: M_Control_6


title: split ePE3 at multiple endogenous sites- Rep 3- I6


title: M_IFN_1


title: M_LPS

value: Retina, 24 hour culture in REM + 0.2ug/mL LPS


title: Healthy_pig_lung1, replicate 1, scRNA-seq

description: 10x Genomics


title: attB/P_mESC_Cre_d3_rep1

description: Library name: SP000253

value: Bl6xCast mESC


title: wt_pig_rep3

city: guangzhou

first: yizhi

last: chen

state: guangdong

address: No.601, West Huangpu Avenue

department: Guangdong-Hong Kong-Macau Institute of CNS Regeneration

institute: Jinan university

library_source: transcriptomic

data_processing:
Raw data (raw reads) of fastq format were firstly processed through Trimmomatic v0.39[2]. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-A and low quality reads from raw data. FastQC v0.11.9[3] software was used to perform quality control on raw data and clean data respectively. Q20, Q30 and GC content were calculated through in-house python scripts. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from Ensembl website directly. Index of the reference genome was built using Hisat2 v2.2.1[4] and clean reads were aligned to the reference genome using Hisat2 v2.2.1 to generate a .sam or .bam files containing the alignments.
The gene expression level quantification was performed separately for each sample using the featureCounts v2.0.1[5] software in the subRead package, and the reads count of each gene was obtained with the parameter "-t exon -g gene_id -s 1". Then results of all samples were combined to obtain the expression matrix of all samples. The RPM (Reads per million mapped reads) of each gene was based on the reads count mapped to this gene and sequencing depth.
Assembly: Sus_scrofa.Sscrofa11.1
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

molecule: polyA RNA

source_name: striatum

extract_protocol:
Total RNAs from striatum of mice, and striatum of pigs were extracted using the Trizol method (Thermo Fisher Scientific, MA, USA).
Sequencing libraries were generated using the SEQUMED ® MustSeq ® 3'mRNA DEG kit（Sequmed，China）.The first step is to capture polyA mRNA with polyT primer and reverse transcript it to form DNA-RNA heterozygous chain. The primer consists of oligo-dT, a sample barcode, a unique and the Illumina-compatible 5ʹ adaptor. Then RNA is removed, the sample is purified with 1X magnetic beads.After the RNA removal,the random primer is used for two-chain synthesis,and the P5 and P7 adaptor that are suitable for the Illumina platform are used for PCR amplification.At last, PCR products were purified 250bp-1000bp fragments for sequencing library.

value: striatum

tag: disease state

value: wt

value: untreated


title: attB/P_mESC_parental_rep1

description: Library name: SP000251


title: M_33C

value: Retina, 24 hour culture in REM at 33 degrees Celcius


title: M_B. Henslae

value: Retina, 24 hour culture in REM + 100CFU/mL B. Henslae


title: wt_pig_rep2


title: M_Control_5

value: retina


title: Shuffle_clone_H12

description: Library name: SP000226


title: M_High Oxygen

value: Retina, 24 hour culture in REM at 40% Oxygen


title: wt_pig_rep1


title: split ePE3 at multiple endogenous sites- Rep 3- I3

value: HEK293T cell


title: Shuffle_clone_G12

description: Library name: SP000225


title: hd_pig_rep3

value: hd


title: M_40C

value: Retina, 24 hour culture in REM at 40 degrees Celcius


title: split ePE3 at multiple endogenous sites- Rep 3- I2


title: M_Control_4

value: Retina, 24 hour culture in REM


title: Shuffle_clone_F12

description: Library name: SP000224


title: Shuffle_clone_D12

description: Library name: SP000222


title: hd_pig_rep2


title: split ePE3 at multiple endogenous sites- Rep 2- I55


title: split ePE3 at multiple endogenous sites- Rep 3- I1


title: casrx_pig_rep3

value: casrx


title: attB/P_mESC_Bxb1_d5_rep2

description: Library name: SP000321


title: M_Low Oxygen_1

value: Retina, 24 hour culture in REM at 2.3% Oxygen


title: M_Kcl

value: Retina, 24 hour culture in REM + 80mM Kcl


title: hd_pig_rep1


title: split ePE3 at multiple endogenous sites- Rep 2- I54


title: Shuffle_clone_C12

description: Library name: SP000221


title: split ePE3 at multiple endogenous sites- Rep 3- I5


title: split ePE3 at multiple endogenous sites- Rep 2- I53


title: casrx_pig_rep2


title: M_Control_3


title: casrx_pig_rep1


title: Shuffle_clone_B12

description: Library name: SP000220


title: split ePE3 at multiple endogenous sites- Rep 2- I51


title: Shuffle_clone_A12

description: Library name: SP000219


title: E9Ctr3

city: Greenville

first: Weihua

last: Huang

state: North Carolina

address: 600 Moye Blvd, Brody 7S-24

department: Pathology

institute: East Carolina University

description: E9.5 embryo

data_processing:
Kallisto pseudoalignment
Assembly: mm10 transcriptome
Supplementary files format and content: counts of each gene in csv format

source_name: Heart

extract_protocol:
Aurum Total RNA Mini Kit
TruSeq Stranded LT mRNA kit

value: Heart

value: C57BL/6 ctrl


title: split ePE3 at multiple endogenous sites- Rep 2- I52


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep2- I5

source_name: Hepa1-6 cell

value: Hepa1-6 cell


title: E9Ctr2

description: E9.5 embryo


title: Shuffle_clone_H11

description: Library name: SP000218

value: Bl6xCast mESC


title: split ePE3 at multiple endogenous sites- Rep 2- I50


title: Shuffle_clone_G11

description: Library name: SP000217


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep3- I6


title: split ePE3 at multiple endogenous sites- Rep 2- I49


title: Shuffle_clone_F11

description: Library name: SP000216


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep2- I2


title: split ePE3 at multiple endogenous sites- Rep 2- I48


title: E9MCKO3

description: E9.5 embryo

value: CDC-42 KO


title: Shuffle_clone_E12

description: Library name: SP000223


title: Shuffle_clone_E11

description: Library name: SP000215


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep3- I3


title: M_Glucose

value: Retina, 24 hour culture in REM + 20mM Glucose


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep1- I4


title: E9Ctr1

description: E9.5 embryo


title: split ePE3 at mPcsk9 in hepa1-6 cells- Rep1- I1


title: Shuffle_clone_D11

description: Library name: SP000214


title: split ePE3 at multiple endogenous sites- Rep 2- I46


title: split ePE3 at multiple endogenous sites- Rep 2- I47


title: E9MCKO1

description: E9.5 embryo


title: Shuffle_clone_C11

description: Library name: SP000213


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 10

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 10
untreat-10

data_processing:
Traget site and protential off-target sites were amplified by PCR using the primer sequences (Table S1), then sequenced on an Illumina NovaSeq system. Deep sequencing data analysis was processed using CRISPResso2 and untreated cells were used as negative controls to exclude PCR amplification and sequencing errors. Reads were align to the amplicon sequence by crispresso2 using --max_paired_end_reads_overlap 140 --min_paired_end_reads_overlap     10 --exclude_bp_from_left 0 --exclude_bp_from_right 0 --plot_window_size 20 --min_frequency_alleles_around_cut_to_plot 0.2
Any aligned reads with a nucleotide sequence within prime editing target window that matches the nucleotide encoded by the pegRNA reverse transcription were noted as off-target reads. Off-target editing efficiencies, which are greater than 0.02%, were quantified as a percentage of (number of off-target reads) / (number of total reads).On-target editing efficiencies, which are greater than 0.1%, were quantified as a percentage of (number of on-target reads) / (number of total reads).
Assembly: EMX1:NNNNAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAACCACAAACCCACGAGGGCAGAG. APP:NNNNTTTGGGTAGGCTTTGTCTTACAGTGTTATTATTTATGAGTAAAACTAATTGGTTGTCCTGCATACTTTAATTATGATGTAATACAGGTTCTGGGTTGACAAATATCAAGACGGAGGAGATCTCTGAAGTGAAGATGGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTACGTAAAATAATTTACCTCTTTCCACTACTGTTTGTCTTGCCAAATGACCTATTAACTCTGGTTCATCCTGTGCTAGAAA. CETP:NNNNTGAAGCTCTGCCTGGGAAGGGCCCACAAGGTGAAATGGGAAGCTCTGTCAGCCTCGGCACCCAGTTTCCCCTCCAGCCCACACTTACGAGACATGACCTCAGGGATGCCCACAGCGGTGATCATTGACTGCAGGAAGCTCTGGACGGACTCGGAGCTGCTCTGCCAATCAAATGCCCATGGTGAGTCCTCCCTGGAGCACAGAAGTGACCCACAAGGCAGATGCTGCACGGCCTGGACAAGCATTCATCCTCATGCCCATCCGTGA. VEGFA:NNNNACACAGCTTCCCGTTCTCAGCTCCACAAACTTGGTGCCAAATTCTTCTCCCCTGGGAAGCATCCCTGGACACTTCCCAAAGGACCCCAGTCACTCCAGCCTGTTGGCTGCCGCTCACTTTGATGTCTGCAGGCCAGATGAGGGCTCCAGATGGCACATTGTCAGAGGGACACACTGTGGCCCCTGTGCCCAGCCCTGGGCTCTCTGTACATGAAGCAACTCCAGTCCCAAATATGTAGCTGTTTGGGAGGTCAGAAATAGGGGGTCCAGGAGCA. ALDOB:NNNNATAGCAGGCTGTGAAGAGCGACTGGGTGGAAGCAGCCCCAGAAGAACCCGTGTGAACATACTGTCCTTTGGCCGCCTGGCAGTTAGCCTAGAAGACAAATATGAGAGAGGAGACTGGTTAGAGTAAATGTGACCCCACCTTGACCCTGTCTGCCTCTCATGGGAAACCAGTCAAACCTTCACTCAGATTTTCTTCTTTTTACAGCAATGTTGCTGAATATTGCTGGGGATACCATCCC. SLC30A8:NNNNGGCTTTGCTTCGTGTCTTCCACTTTTACCCTTGGTTTGACTTTGCAGCACCAGCCTGCTGTTATTGCTCACTCTATGAACCGTACCTGCTCGGTGCCATCCAAATGTCAGCCGCTTAGAGGGAGGCTTCGATGACAACCACAGGGAGAAGAGACTGAGCAGGAAACTGGTCAGGTCAATTAAGAGGTGGGCAGCATCTGTGACAACAGCAAGACTCCCAGCAATGTGCCCACCTAGAGAATGCAAAATAGTACACTGTGTTAAGGTCCCAGAATGCAGCTAGATTCAC. MECP2:NNNNGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGGCCACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGG. RNF2:NNNNAAAGTGTTAGCCAACATACAGAAGTCAGGAATGCTTGAATATAAATTTATTATTACTCTATGTTCTATTTAAGTTTTCATGTTCTAAAAATGTATCCCAGTTTACACGTCTCATATGCCCCTTGGCAGTCATCTTAGTCATTACCTGAGGTGTTCGTTGTAACTCATATAAACTGAGTTCCCATGTTTTGCTTAATGGTTGAGTTCCGTTTGTCTGCAC. HEK3:NNNNGATGTGGGCTGCCTAGAAAGGCATGGATGAGAGAAGCCTGGAGACAGGGATCCCAGGGAAACGCCCATGCAATTAGTCTATTTCTGCTGCAAGTAAGCATGCATTTGTAGGCTTGATGCTTTTTTTCTGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCCCAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAGGGCGTCGCAGGACAGCTTTTCCTAGACAGGGGCT. ANGPTL3:NNNNTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATC. TTR:NNNNGCAGCCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAGGATGGCTTCTCATCGTCTGCTCCTCCTCTGCCTTGCTGGACTGGTATTTGTGTCTGAGGCTGGCCCTACGGTGAGTGTTTCTGTGACATCCCATTCCTACATTTAAGATTCACGCTAAATGAAGTAGAAGTGACTCCTTCCAGCTTTGCCAACCAGCTTTTATTACTAGGGCAAGGGTACCCAG. FANCF:NNNNTCGCGGATGTTCCAATCAGTACGCAGAGAGTCGCCGTCTCCAAGGTGAAAGCGGAAGTAGGGCCTTCGCGCACCTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGGCGGTCTCAAGCACTACCTACGTCAGCACCTGGGACCCCGCCACCGTGCGCCGGGCCTTGCAGTGGGCGCGCTACCTGCGCCACATCCATCGGCGCTTTGGTCGGCATGGCCCCATTCGCACGGCTCTGGA. mPcsk9:NNNNTGGGGAGGTGGACAGTCAGGTGGGGCTGCTGTGGGCTGCTATTAGCTGAAGGGCTTTTGAAGCTAAGTGCATGGCTGTCTGGTTCTGTAGGCCCTGAAGTTGCCCCATGTGGAGTACATTGAGGAAGACTCCTTTGTCTTCGCCCAGAGCATCCCATGGAACCTGGAGCGAATTATCCCAGCATGGCACCAGACAGAGGAAGACCGCTCCCCTGGTAAGGTCCCCAGTAAGCCTCCAGCCTCCACTTGCTTTGCTCTGCCCGTCCTCTTCCCAA.
Supplementary files format and content: xlsx files,percentage of total sequencing reads with on-target editing or off-target editing.
Library strategy: Amplicon Sequencing

source_name: liver

growth_protocol: All animal experiments were conducted under the protocol approved by the Animal Care and  Ethical Committee of the Westlake University. C57BL/6 and ICR mice were purchased and  housed in the Laboratory Animal Resource Center (LARC) at the Westlake University. The  LARC is a certified pathogen-free and environmental-control facility (21±2°C, 55±15%  humidity and 12:12-h light : dark cycle).

extract_protocol:
After three weeks, the mice were euthanized using carbon dioxide, and their  livers were dissected. The livers were washed thoroughly with pre-chilled PBS, minced with  surgical scissors, and placed in 2 mL sterile Eppendorf tubes. Two 4 mm and two 2 mm  stainless steel beads were added to each tube. The tissues were homogenized using the Qiagen Tissuelyser II at a power setting of 30 for 3 minutes, followed by reversing the adapter  orientation and homogenizing again at the same power setting for an additional 3 minutes.  Portions of the homogenized tissue were used for DNA extraction. Genomic DNA (gDNA)  from the organs was extracted using the TIANamp Genomic DNA Kit (TIANGEN  #DP304-03) according to the manufacturer’s instructions.
Use site-specific primers with second-generation sequencing adapters to amplify on-target and off-target sites via PCR for library construction.

treatment_protocol: AAVwasdiluted into pre-chilled PBS to a concentration of 2 × 1013/ml. Subsequently, 125 μl  of CBH608 and 125 μl of CBH621 were mixed thoroughly. Using a sterile 0.6 × 25 mm  TWLBsyringe, 200 μl of the mixture containing 1 × 1012 of each virus, were injected into the  tail vein of six-week-old male C57BL/6 mice. This experiment was repeated three times  independently

value: liver

value: untreat


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 9

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 9
untreat-9


title: Shuffle_clone_A11

description: Library name: SP000211


title: split ePE3 at multiple endogenous sites- Rep 2- I43


title: split ePE3 at multiple endogenous sites- Rep 2- I45


title: Shuffle_clone_B11

description: Library name: SP000212


title: RAW 264.7 cells, siLCN, Day 4_3

city: Seoul

first: Heeeun

last: Woo

address: 1, Anam-dong 5(o)-ga, Seongbuk-gu

country: South Korea

institute: Korea University

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and filtered for low-complexity or low-quality sequence(using fastp), then mapped to GRCm39 (NCBI) whole genome using STAR.
Read count extraction using HTSeq-count.
Assembly: GRCm39
Supplementary files format and content: tab-delimited text files include read count values for each Sample

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). 500 ng of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using LEXOGEN Quant-Seq Library Prep Kit (Cat#015) standard protocols.

value: macrophage

tag: treatment 1

value: Lcn2 siRNA

tag: treatment 2

value: osteoclast differentiation

tag: time

value: Day 4


title: RAW 264.7 cells, siLCN, Day 4_2


title: E9MCKO2

description: E9.5 embryo


title: RAW 264.7 cells, CPN, Day 4_3

value: chaperonin


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 6

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 6
untreat-6


title: split ePE3 at multiple endogenous sites- Rep 2- I44

value: HEK293T cell


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 5

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 5
untreat-5


title: Shuffle_clone_H10

description: Library name: SP000210


title: split ePE3 at multiple endogenous sites- Rep 2- I39


title: RAW 264.7 cells, control, Day 4_3

value: normal control


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 7

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 7
untreat-7


title: Shuffle_clone_E10

description: Library name: SP000207


title: split ePE3 at multiple endogenous sites- Rep 2- I42


title: Shuffle_clone_D10

description: Library name: SP000206


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 8

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 8
untreat-8


title: Shuffle_clone_F10

description: Library name: SP000208


title: Shuffle_clone_G10

description: Library name: SP000209


title: split ePE3 at multiple endogenous sites- Rep 2- I41


title: RAW 264.7 cells, CPN, Day 4_1


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 4

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 4
untreat-4


title: RAW 264.7 cells, CPN, Day 4_2


title: split ePE3 at multiple endogenous sites- Rep 2- I40


title: RAW 264.7 cells, control, Day 4_1


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 2

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 2
untreat-2


title: Shuffle_clone_C10

description: Library name: SP000205


title: Shuffle_clone_B10

description: Library name: SP000204


title: split ePE3 at multiple endogenous sites- Rep 2- I36


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 3

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 3
untreat-3


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 10

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 10
R3-10

value: In vivo prime editing to install Pcsk9 Q155H with dual-AAV split-ePE3-369


title: split ePE3 at multiple endogenous sites- Rep 2- I38


title: RAW 264.7 cells, siLCN, Day 4_1


title: Shuffle_clone_A10

description: Library name: SP000203


title: IPP_120hpf_brain_replicate4

city: Clemson

first: Subham

last: Dasgupta

state: South Carolina

address: 142 Long Hall

department: Biological Sciences

institute: Clemson University

description:
IPP_br_4
gene_fpkm_brain.txt

data_processing:
Raw data (raw reads) in fastq format was firstly processed throughin-house perl scripts. In this step, clean data (clean reads) was obtainedbyremoving reads containing adapter, reads containing ploy-N and lowqualityreads from raw data. A
Reference genome and gene model annotation files were downloadedfrom genome website directly. Index of the reference genome was built usingHisat2 v2.0.5 and paired-end clean reads were aligned to the referencegenome using Hisat2 v2.0.5
FeatureCounts v1.5.0-p3 was used to count the reads numbers mappedto each gene. Then FPKM of each gene was calculated based on the lengthof the gene and reads count mapped to this gene.
Differential expression analysisof two conditions/groups (two biological replicates per condition) was performed using the DESeq2R package (1.20.0).
Assembly: GRZ11
Supplementary files format and content: Tab-delimited txt file contains FPKM values for each sample

organism: Danio rerio

source_name: Dissected brain

growth_protocol: Embryos were spawned and maintained in system water

extract_protocol:
Eyes and brains were dissected from whole embryos, homogenized in  RNA zol, and RNA extraction was conducted using a Directzol RNA Miniprep Plus kit (Zymo). RNA quality and quantity was validated using an Agilent Tapestation and Nanodrop.
After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library.
For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification(
For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification(

treatment_protocol: Embryos were exposed to 0 or 0.2 uM IPP in 0.02% DMSO from 6 to 120 hpf

value: Dissected brain

value: wildtype

value: IPP


title: split ePE3 at multiple endogenous sites- Rep 3- I4


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,off target 1

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, untreat control,off target 1
untreat-1


title: split ePE3 at multiple endogenous sites- Rep 2- I37


title: Shuffle_clone_G9

description: Library name: SP000201


title: IPP_120hpf_brain_replicate3

description:
IPP_br_3
gene_fpkm_brain.txt


title: Shuffle_clone_H9

description: Library name: SP000202


title: split ePE3 at multiple endogenous sites- Rep 2- I35


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 7

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 7
R3-7


title: split ePE3 at multiple endogenous sites- Rep 2- I34


title: IPP_120hpf_brain_replicate2

description:
IPP_br_2
gene_fpkm_brain.txt


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 8

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 8
R3-8


title: split ePE3 at multiple endogenous sites- Rep 2- I33


title: RAW 264.7 cells, control, Day 4_2


title: IPP_120hpf_brain_replicate1

description:
IPP_br_1
gene_fpkm_brain.txt


title: Shuffle_clone_D9

description: Library name: SP000198


title: Shuffle_clone_F9

description: Library name: SP000200


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 9

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 9
R3-9


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 6

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 6
R3-6


title: split ePE3 at multiple endogenous sites- Rep 2- I30


title: Shuffle_clone_C9

description: Library name: SP000197


title: split ePE3 at multiple endogenous sites- Rep 2- I29


title: DMSO_120hpf_brain_replicate3

description:
Ctrl_br_3
gene_fpkm_brain.txt

value: DMSO


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 5

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 5
R3-5


title: split ePE3 at multiple endogenous sites- Rep 2- I31


title: split ePE3 at multiple endogenous sites- Rep 2- I28


title: DMSO_120hpf_brain_replicate1

description:
Ctrl_br_1
gene_fpkm_brain.txt


title: Shuffle_clone_E9

description: Library name: SP000199

value: Bl6xCast mESC


title: DMSO_120hpf_brain_replicate2

description:
Ctrl_br_2
gene_fpkm_brain.txt


title: IPP_120hpf_eye_replicate4

description:
IPP_eye_4
gene_fpkm_eye.txt

source_name: Dissected eye

value: Dissected eye


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 4

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 4
R3-4


title: Shuffle_clone_A9

description: Library name: SP000195


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 3

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 3
R3-3


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 2

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 2
R3-2


title: IPP_120hpf_eye_replicate3

description:
IPP_eye_3
gene_fpkm_eye.txt


title: split ePE3 at multiple endogenous sites- Rep 2- I27


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,off target 1

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate3, off target 1
R3-1


title: Shuffle_clone_H8

description: Library name: SP000194


title: IPP_120hpf_eye_replicate2

description:
IPP_eye_2
gene_fpkm_eye.txt


title: Shuffle_clone_B9

description: Library name: SP000196


title: DMSO_120hpf_brain_replicate4

description:
Ctrl_br_4
gene_fpkm_brain.txt


title: split ePE3 at multiple endogenous sites- Rep 2- I26


title: split ePE3 at multiple endogenous sites- Rep 2- I25


title: split ePE3 at multiple endogenous sites- Rep 2- I32


title: IPP_120hpf_eye_replicate1

description:
IPP_eye_1
gene_fpkm_eye.txt


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 10

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 10
R2-10


title: Shuffle_clone_E8

description: Library name: SP000191


title: Shuffle_clone_F8

description: Library name: SP000192


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 9

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 9
R2-9


title: split ePE3 at multiple endogenous sites- Rep 2- I23


title: split ePE3 at multiple endogenous sites- Rep 2- I24


title: DMSO_120hpf_eye_replicate3

description:
Ctrl_eye_3
gene_fpkm_eye.txt


title: DMSO_120hpf_eye_replicate4

description:
Ctrl_eye_4
gene_fpkm_eye.txt


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 8

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 8
R2-8


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 7

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 7
R2-7


title: Shuffle_clone_D8

description: Library name: SP000190


title: DMSO_120hpf_eye_replicate2

description:
Ctrl_eye_2
gene_fpkm_eye.txt


title: split ePE3 at multiple endogenous sites- Rep 2- I21


title: split ePE3 at multiple endogenous sites- Rep 2- I20


title: DMSO_120hpf_eye_replicate1

description:
Ctrl_eye_1
gene_fpkm_eye.txt


title: Shuffle_clone_B8

description: Library name: SP000188


title: Shuffle_clone_C8

description: Library name: SP000189


title: split ePE3 at multiple endogenous sites- Rep 2- I22


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 6

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 6
R2-6


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 5

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 5
R2-5


title: pigpbmc1

city: Beijing

first: Zhaoqi

last: Zhang

state: Beijing

address: Beijing China Institute of Zoology，Chinese Academy of Sciences

institute: University of Chinese Academy of Sciences

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Supplementary files format and content: GRCh38 and susScr11_genome_10.2
Supplementary files format and content: Cell Ranger output files (barcodes.tsv, features.tsv, matrix.mtx)

source_name: blood

extract_protocol:
The pig is a promising candidate for xenotransplantation. Understanding the differences between human and swine immune systems is critical for addressing xeno-transplant rejection and hematopoietic reconstitution. We performed single-cell RNA-seq on human and pig immune cells. High oxidative phosphorylation, HIF-1, glycolysis, and lysosome-related gene expressions in pig CD14+ monocytes were observed, whereas pig CD14+ monocytes exhibited lower levels of cytokine receptors and JAK-STAT-related genes. Pig activated CD4+T cells decreased cell adhesion and inflammation, while enriched for migration and activation processes. Porcine GNLY+CD8+T cells reduced cytotoxicity and increased proliferation compared with human GNLY+CD8+T cells. Pig CD2+CD8+γδT cells were functionally homologous to human CD2+CD4+ γδT cells. Pig CD2-CD8-γδT cells expressed genes with quiescent and precursor characteristics, while CD2-CD8+γδT cells expressed migration and memory-related molecules. Pig CD24+ and CD5+B cells are associated with inflammatory responses. Our research with integrated scRNA-seq enables a deeper understanding of the development and function of porcine immune cells.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

value: blood

value: PBMC


title: split ePE3 at multiple endogenous sites- Rep 2- I19


title: Shuffle_clone_H7

description: Library name: SP000186


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 3

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 3
R2-3

value: liver


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 4

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 4
R2-4


title: Shuffle_clone_A8

description: Library name: SP000187


title: Shuffle_clone_G7

description: Library name: SP000185


title: pigpbmc2

description: 10x Genomics


title: split ePE3 at multiple endogenous sites- Rep 2- I18


title: SA_O_06_34_Lu

city: Morgantown

first: Pius

last: Joseph

address: 1000 Frederick Lane

institute: NIOSH

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_34_Lu

data_processing:
Basecalls were performed using bcl2fastq/2.19.
Sequence reads were trimmed using Trim Galore!, Cutadapt 3.4 and Trimgalore 0.6.7 using Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
All sequences were aligned to the Rattus norvegicus Rnor 6.0 genome from Ensembl downloaded from igenomes using Star 2.6.1d within  Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
Raw gene counts were assigned using Salmon 1.10.1 and Sammtools 1.17 within Nextflow 22.10.6 and the nf-core/rnaseq 3.12.0 pipeline.
Assembly: Rnor6.0
Supplementary files format and content: tsv file including abundance measurements (raw counts)

organism: Rattus norvegicus

source_name: lung

growth_protocol: Healthy male Fischer 344 rats (Charles River Wilmington, MA), weighing 250-300 g were used in this study which was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International approved animal facility (NIOSH, Morgantown, WV) following a protocol approved by the Animal Care and Use Committee. The rats were housed in pairs/cage and maintained on a 12-hour light-dark cycle in a temperature (68-72oF) and humidity (30-70%) controlled room with free access to filtered tap water and HaCXan Teklad Global 18% protein rodent diet (Harlan Teklad; Madison, WI).

extract_protocol:
Total RNA from the lungs were isolated using the RNeasy Fibrous Mini Kit (Qiagen, Inc, Valencia, CA). RNA from whole blood, collected in K2EDTA BD Vacutainer™ tubes (Becton Dickinson; Franklin Lakes, NJ) and then stored 1 part blood in 3 parts RNALater (ThermoFisher Scientific; Waltham, MA), was isolated using the mouse Ribopure RNA Isolation Kit (Ambion AM1951) with optional DNase treatment using the Invitrogen™ DNA-free™ DNA Removal Kit (Invitrogen by ThermoFisher Scientific; Waltham, MA). Immediately following RNA isolation and DNase treatment, RNA samples were depleted of alpha and beta globin mRNAs using the Ambion® GLOBINclear™-Mouse/Rat Kit, as directed by the manufacturer’s protocol.
Next generation sequencing libraries prepared using TruSeq® Stranded mRNA Library Prep Kit (Illumina, Inc, San Diego, CA) were used in the sequencing.

treatment_protocol: The rats were exposed by whole-body inhalation to filtered air or crystalline silica (Min-U-Sil 5) (15 mg/m3, 6 hours/day, 5 days).

value: lung

value: Fischer 344

tag: time point

value: 6 months

value: SILICA


title: split ePE3 at multiple endogenous sites- Rep 2- I17


title: SA_O_06_33_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_33_Lu


title: SA_O_06_32_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_32_Lu


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 10

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 10
R1-10


title: Shuffle_clone_E7

description: Library name: SP000183


title: SA_O_06_31_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_31_Lu


title: Shuffle_clone_D7

description: Library name: SP000182


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 9

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 9
R1-9


title: SA_O_06_35_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_35_Lu


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 1

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 1
R2-1


title: split ePE3 at multiple endogenous sites- Rep 2- I14


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,off target 2

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate2, off target 2
R2-2


title: Shuffle_clone_F7

description: Library name: SP000184


title: split ePE3 at multiple endogenous sites- Rep 2- I15


title: split ePE3 at multiple endogenous sites- Rep 2- I13


title: split ePE3 at multiple endogenous sites- Rep 2- I16


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 7

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 7
R1-7


title: split ePE3 at multiple endogenous sites- Rep 2- I10


title: SA_O_06_30_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_30_Lu


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 8

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 8
R1-8


title: split ePE3 at multiple endogenous sites- Rep 2- I11


title: SA_O_06_25_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_25_Lu

value: AIR


title: split ePE3 at multiple endogenous sites- Rep 2- I12

value: HEK293T cell


title: Shuffle_clone_B7

description: Library name: SP000180


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 4

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 4
R1-4


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 6

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 6
R1-6


title: Shuffle_clone_A7

description: Library name: SP000179


title: Shuffle_clone_H6

description: Library name: SP000178


title: split ePE3 at multiple endogenous sites- Rep 2- I9


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 5

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 5
R1-5


title: SA_O_06_24_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_24_Lu


title: Shuffle_clone_F6

description: Library name: SP000176


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 3

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 3
R1-3


title: split ePE3 at multiple endogenous sites- Rep 2- I8


title: SA_O_01_14_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_14_Lu

value: 1 day


title: SA_O_06_20_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_20_Lu


title: Shuffle_clone_E6

description: Library name: SP000175


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 1

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 1
R1-1

value: In vivo prime editing to install Pcsk9 Q155H with dual-AAV split-ePE3-369


title: SA_O_06_19_Lu

description:
salmon.merged.gene_counts_SAO_Lu06.tsv
SA_O_06_19_Lu


title: Shuffle_clone_G6

description: Library name: SP000177


title: split ePE3 at multiple endogenous sites- Rep 2- I6


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,off target 2

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue,  replicate1, off target 2
R1-2


title: SA_O_01_15_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_15_Lu


title: Shuffle_clone_C6

description: Library name: SP000173


title: split ePE3 at multiple endogenous sites- Rep 2- I7


title: Shuffle_clone_B6

description: Library name: SP000172


title: split ePE3 at multiple endogenous sites- Rep 2- I4


title: SA_O_01_11_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_11_Lu


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep2,on target

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, on target, replicate2
0621_2

value: In vivo prime editing to install Pcsk9 Q155H with dual-AAV split-ePE3-368


title: split ePE3 at multiple endogenous sites- Rep 2- I3


title: Shuffle_clone_C7

description: Library name: SP000181


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,untreat,on target

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, on target, untreat control
untreat-on


title: Shuffle_clone_H5

description: Library name: SP000170


title: SA_O_01_12_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_12_Lu


title: Shuffle_clone_A6

description: Library name: SP000171


title: Shuffle_clone_D6

description: Library name: SP000174


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep1,on target

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, on target, replicate1
0621_1

value: In vivo prime editing to install Pcsk9 Q155H with dual-AAV split-ePE3-367


title: liver,dual-AAV split-ePE3-367, tail-vein injection,liver,rep3,on target

description:
In vivo prime editing to install Pcsk9 Q155H by split-ePE3-367 at liver tissue, on target, replicate3
0621_3


title: SA_O_01_08_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_08_Lu


title: Shuffle_clone_G5

description: Library name: SP000169


title: SA_O_01_10_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_10_Lu


title: split ePE3 at multiple endogenous sites- Rep 2- I1


title: SA_O_01_13_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_13_Lu


title: cells with Relma, IAV infection, rep4

city: Riverside

first: Brandon

last: Le

state: CA

address: 3401 Watkins Drive

department: Institute for Integrative Genome Biology

institute: University of California, Riverside

data_processing:
Raw data were pre-processed for quality control using Fastqc (v 0.11.9) and Trim_galore (v 0.6.7).
Processed reads were aligned the mouse reference genome (GRCm39) using STAR aligner (v.27.9a) with parameters --outFilterMismatchNmax 0 and --outFilterMultimapNmax 1.
Read counts were quantified using Featurecounts (v2.0.3).
Assembly: GRCm39
Supplementary files format and content: A tab-delimited text file containing raw counts for each sample (columns)

growth_protocol: MLE-12 cells were cultured in HITES Media (DMEM/F-12 with HEPES, 1X Insulin-Transferrin-Selenium, 2% FBS, 100U/ml Penicillin-Streptomycin, 2mM L-glutamine (Gibco), 10 nM Hydrocortisone, 10nM β-estradiol (Sigma-Aldrich) at 37°C in 5% CO2.

extract_protocol:
Cells were recovered 24 hours post infection and subjected to RNA isolation using the Promega total RNA isolation kit (Promega, Cat#Z3100)
Total RNA were sent to Novogene, Inc for library construction and sequencing

treatment_protocol: MLE-12 cells were treated with RELMα (500ng/mL) and infected one hour later with IAV.

value: epithelial cell

value: RELMa

tag: infection

value: IAV infection

tag: biological replicate


title: Shuffle_clone_F5

description: Library name: SP000168

value: Bl6xCast mESC


title: split ePE3 at multiple endogenous sites- Rep 2- I5


title: cells with Relma, IAV infection, rep3


title: SA_O_01_06_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_06_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I55


title: SA_O_01_05_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_05_Lu


title: Shuffle_clone_E5

description: Library name: SP000167


title: cells with Relma, IAV infection, rep2


title: Shuffle_clone_G8

description: Library name: SP000193


title: Shuffle_clone_D5

description: Library name: SP000166


title: SA_O_01_04_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_04_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I53


title: cells with PBS, IAV infection, rep4

value: PBS


title: cells with Relma, IAV infection, rep1


title: SA_O_01_02_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_02_Lu


title: Shuffle_clone_C5

description: Library name: SP000165


title: split ePE3 at multiple endogenous sites- Rep 1- I52


title: Shuffle_clone_B5

description: Library name: SP000164


title: SA_O_06_36_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_36_BL

value: blood


title: cells with PBS, IAV infection, rep3


title: SA_O_01_07_Lu

description:
salmon.merged.gene_counts_SAO_Lu01.tsv
SA_O_01_07_Lu


title: cells with PBS, IAV infection, rep2


title: split ePE3 at multiple endogenous sites- Rep 2- I2


title: split ePE3 at multiple endogenous sites- Rep 1- I51


title: Shuffle_clone_A5

description: Library name: SP000163


title: SA_O_06_34_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_34_BL


title: Shuffle_clone_H4

description: Library name: SP000162


title: SA_O_06_33_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_33_BL


title: cells with PBS, IAV infection, rep1


title: Shuffle_clone_G4

description: Library name: SP000161


title: split ePE3 at multiple endogenous sites- Rep 1- I49


title: split ePE3 at multiple endogenous sites- Rep 1- I54


title: Shuffle_clone_F4

description: Library name: SP000160


title: split ePE3 at multiple endogenous sites- Rep 1- I48


title: cells with Relma, no infection, rep4

value: no infection


title: cells with Relma, no infection, rep3

value: lung


title: SA_O_06_31_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_31_BL

value: Fischer 344


title: cells with Relma, no infection, rep2


title: split ePE3 at multiple endogenous sites- Rep 1- I46


title: SA_O_06_30_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_30_BL

value: 6 months


title: Shuffle_clone_D4

description: Library name: SP000158


title: Shuffle_clone_C4

description: Library name: SP000157


title: SA_O_06_27_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_27_BL


title: cells with PBS, no infection, rep4


title: split ePE3 at multiple endogenous sites- Rep 1- I50


title: Shuffle_clone_B4

description: Library name: SP000156


title: split ePE3 at multiple endogenous sites- Rep 1- I44


title: cells with PBS, no infection, rep3


title: split ePE3 at multiple endogenous sites- Rep 1- I45


title: SA_O_06_25_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_25_BL


title: SA_O_06_23_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_23_BL


title: Shuffle_clone_A4

description: Library name: SP000155


title: SA_O_06_32_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_32_BL

value: SILICA


title: cells with PBS, no infection, rep2


title: split ePE3 at multiple endogenous sites- Rep 1- I47


title: split ePE3 at multiple endogenous sites- Rep 1- I41


title: SA_O_06_20_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_20_BL

value: AIR


title: Shuffle_clone_G3

description: Library name: SP000153


title: SA_O_06_21_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_21_BL


title: SA_O_06_19_BL

description:
salmon.merged.gene_counts_SAO_BL06.tsv
SA_O_06_19_BL


title: cells with PBS, no infection, rep1


title: Neuro2a_D-Ser_3

city: Tokyo

first: Jumpei

last: Sasabe

address: 35 Shinanomachi, Shinjuku

department: Pharmacology

country: Japan

institute: Keio University School of Medicine

data_processing:
Paired end sequences were read by NovaSeq (Illumina).
BCL/cBCL (base call) binary files are converted into GASTQ files using bcl2fastq.
Adapter sequences were trimmed from raw sequences with Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and trimmed sequences were mapped to mouse genome (GRCm38/mm10) using HISAT2 (https://daehwankimlab.github.io/hisat2/).
Aligned sequences were counted using FeatureCounts (https://subread.sourceforge.net/). Differentially expressed genes were identified by DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html).
Assembly: GRCm38/mm10
Supplementary files format and content: csv file containing matrix of read counts data per gene in row and sample in column.

source_name: cell line

growth_protocol: Neuro2a cells were cultured in D-MEM with 10% FBS.

extract_protocol:
Cells were harvested in Trizol and RNA was extracted from cells according to the manufacture’s protocol.
mRNA libraries of each samples were prepared using a TruSeq Stranded mRNA library Kit (Illumina, San Diego, CA) according to the manufactures’ protocol  (TruSeq Stranded mRNA Sample Preparation Guide, Part # 15031047 Rev. E)

treatment_protocol: The medium was changed to D-MEM(-SG) including 10% FBS with or without 5 mM D-serine for 18 hours.

value: cell line

value: mouse neuroblastoma

value: 5mM D-serine


title: split ePE3 at multiple endogenous sites- Rep 1- I40

value: HEK293T cell


title: cells with Relma, no infection, rep1


title: Shuffle_clone_H3

description: Library name: SP000154


title: Shuffle_clone_E4

description: Library name: SP000159


title: SA_O_01_18_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_18_BL

value: 1 day


title: Neuro2a_D-Ser_2


title: split ePE3 at multiple endogenous sites- Rep 1- I39


title: Shuffle_clone_E3

description: Library name: SP000151


title: Shuffle_clone_F3

description: Library name: SP000152


title: SA_O_01_16_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_16_BL


title: Neuro2a_D-Ser_1


title: split ePE3 at multiple endogenous sites- Rep 1- I38


title: split ePE3 at multiple endogenous sites- Rep 1- I43


title: SA_O_01_15_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_15_BL


title: Shuffle_clone_D3

description: Library name: SP000150


title: Neuro2a_control_3

value: vehicle


title: split ePE3 at multiple endogenous sites- Rep 1- I37


title: split ePE3 at multiple endogenous sites- Rep 1- I42


title: Neuro2a_control_2


title: split ePE3 at multiple endogenous sites- Rep 1- I36


title: SA_O_01_14_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_14_BL


title: Shuffle_clone_B3

description: Library name: SP000148


title: Neuro2a_control_1


title: Shuffle_clone_H2

description: Library name: SP000146


title: SA_O_01_11_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_11_BL


title: VEEV + CCI mouse 4

city: Blacksburg

first: Alicia

last: Pickrell

address: 970 Washington St NW

institute: Virginia Tech

data_processing:
Bases with quality scores less than 30 and adapters were trimmed from raw sequencing reads by Trim Galore (v 0.6.4)
After trimming, only reads with length greater than 30bp were mapped to mm10 by STAR (v 2.7.1a).
Raw counts and normalized counts for each gene were outputted by RSEM (v1.2.28).
The raw counts were used to identify differentially expressed genes by DESeq2 (v 1.36.0)
Assembly: mm10
Supplementary files format and content: tab-delimited text files include raw count and TPM values for each Sample

source_name: hippocampus

extract_protocol:
Trizol/Directzol/Qiagen
RNA-seq libraries were prepared following the Illumina TruSeq stranded mRNA kit and sequenced using the NovaSeq platform by MedGenome

value: hippocampus

tag: group

value: VEEV + CCI


title: SA_O_01_09_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_09_BL


title: SA_O_01_12_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_12_BL


title: VEEV + CCI mouse 3


title: Shuffle_clone_C3

description: Library name: SP000149

source_name: Bl6xCast mESC

value: Bl6xCast mESC


title: VEEV + CCI mouse 1


title: Shuffle_clone_A3

description: Library name: SP000147


title: Shuffle_clone_E2

description: Library name: SP000143


title: split ePE3 at multiple endogenous sites- Rep 1- I34


title: Shuffle_clone_G2

description: Library name: SP000145


title: SA_O_01_08_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_08_BL


title: VEEV mouse 3

value: VEEV


title: split ePE3 at multiple endogenous sites- Rep 1- I32


title: split ePE3 at multiple endogenous sites- Rep 1- I33


title: SA_O_01_04_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_04_BL

value: blood


title: Shuffle_clone_F2

description: Library name: SP000144


title: SA_O_01_07_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_07_BL


title: split ePE3 at multiple endogenous sites- Rep 1- I31


title: VEEV mouse 4


title: split ePE3 at multiple endogenous sites- Rep 1- I29


title: VEEV mouse 2


title: Shuffle_clone_C2

description: Library name: SP000141


title: SA_O_01_02_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_02_BL


title: SA_O_01_05_BL

description:
salmon.merged.gene_counts_SAO_BL01.tsv
SA_O_01_05_BL


title: VEEV + CCI mouse 2


title: VEEV mouse 1


title: Shuffle_clone_D2

description: Library name: SP000142


title: split ePE3 at multiple endogenous sites- Rep 1- I27


title: split ePE3 at multiple endogenous sites- Rep 1- I35


title: SA-Y-06-62_Lu

value: male


title: split ePE3 at multiple endogenous sites- Rep 1- I30


title: split ePE3 at multiple endogenous sites- Rep 1- I26


title: mock mouse 3

value: mock


title: Shuffle_clone_B2

description: Library name: SP000140


title: split ePE3 at multiple endogenous sites- Rep 1- I28


title: SA-Y-06-60_Lu

value: lung

value: Fischer 344


title: split ePE3 at multiple endogenous sites- Rep 1- I24


title: mock mouse 1


title: SA-Y-06-64_Lu

value: 6 months

value: SILICA


title: Shuffle_clone_F1

description: Library name: SP000136


title: Shuffle_clone_A2

description: Library name: SP000139


title: split ePE3 at multiple endogenous sites- Rep 1- I23


title: mock mouse 4


title: SA-Y-06-58_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I22


title: M_B.Hen_bio_rep_2

data_processing:
Read alignment to the reference genome was performed with STAR (version 2.7; PMID: 23104886) software. Gene level quantification was determined using RSEM (version 1.3.1 ; PMID: 21816040).
Assembly: mm10-mouse / GRCh38 – human
Supplementary files format and content: tab delimited text file with raw counts for each sample

extract_protocol:
Retinal punches were dissolved in 800uL Trizol Reagent (Invitrogen #15596026). 200uL chloroform was added to each sample, inverted, incubated at room temperature for 3 minutes. Samples were then spun down at 12,000 3 g at 4 C for 15 min. The aqueous later was transferred to a new tube and 1uL glycogen and 500 uL isopropanol was added to each sample and mixed by vortex. Samples were incubated at room temperature for 10 minutes and spun down at 12,000 3 g at 4 C for 15 min. The isopropanol was removed and the pellet was washed twice with ice-cold 80% EtOH. The washed pellet was resuspened in water.
Libraries were constructed using Illumina library prep acording to manufacturer protocols

value: retina

value: 24 hour culture in REM + 100CFU/mL B. Henslae


title: M_B.Hen_bio_rep_1


title: Shuffle_clone_H1

description: Library name: SP000138


title: split ePE3 at multiple endogenous sites- Rep 1- I21

data_processing:
Traget site and protential off-target sites were amplified by PCR using the primer sequences (Table S1), then sequenced on an Illumina NovaSeq system. Deep sequencing data analysis was processed using CRISPResso2 and untreated cells were used as negative controls to exclude PCR amplification and sequencing errors. Reads were align to the amplicon sequence by crispresso2 using --max_paired_end_reads_overlap 140 --min_paired_end_reads_overlap     10 --exclude_bp_from_left 0 --exclude_bp_from_right 0 --plot_window_size 20 --min_frequency_alleles_around_cut_to_plot 0.2
On-target editing efficiencies, which are greater than 0.1%, were quantified as a percentage of (number of on-target reads) / (number of total reads).
Supplementary files format and content: xlsx files, percentage of total sequencing reads with on-target editing for all libraries, percentage of total sequencing reads with indels for ePE3-hepa.
Library strategy: Amplicon Sequencing

extract_protocol:
Genomic DNA (gDNA) was isolated using TIANamp Genomic DNA Kit (TIANGEN #DP304) according to the manufacturer’s protocol.
The region that integrated with genomic DNA including RT template was PCR amplified from genomic DNA for HTS. For the first round of PCR, genomic sites of interest were amplified from 150 ng of genomic DNA using Q5 PCR Master Mix (New England Biolabs #M0544S) with the primers containing Illumina forward and reverse adapters. 50 uL of PCR 1 reaction was performed with forward and reverse primers each at 0.25 uM, ~1 uL of genomic DNA extract, and 25 uL of Q5 PCR Master Mix. To add a unique sequencing index, 50 uL of a given PCR 2 contained 2.5 uL of previous step unpurified PCR product; a unique forward or reverse primer pair, each at 0.25 uM; and 25 uL of Q5 PCR Master Mix. Primers are listed in Table S1. PCR 2 products were purified by DNA AMPure XP beads (Beckman Coulter #A63882) and eluted with 25 uL of water. DNA concentration was quantified by Nanodrop One (Thermo Fisher Scientific).


title: Shuffle_clone_C1

description: Library name: SP000133


title: mock mouse 2


title: Shuffle_clone_G1

description: Library name: SP000137


title: SA-Y-06-59_Lu


title: SA-Y-06-61_Lu


title: Shuffle_clone_E1

description: Library name: SP000135


title: Shuffle_clone_D1

description: Library name: SP000134


title: M_Control_bio_rep_6

value: 24 hour culture in REM


title: split ePE3 at multiple endogenous sites- Rep 1- I19

value: HEK293T cell


title: Shuffle_clone_A1

description: Library name: SP000131


title: SA-Y-06-54_Lu

value: AIR


title: split ePE3 at multiple endogenous sites- Rep 1- I18


title: M_Control_bio_rep_7


title: SA-Y-06-53_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I25


title: Cre_Pifi48h-at24h_d5_rep1

description: Library name: SP000116


title: Shuffle_clone_B1

description: Library name: SP000132


title: split ePE3 at multiple endogenous sites- Rep 1- I20


title: SA-Y-06-52_Lu


title: M_2.3%O2_bio_rep_2

value: 24 hour culture in REM at 2.3% Oxygen


title: SA-Y-01-48_Lu

value: 1 day


title: M_2.3%O2_bio_rep_1


title: Cre_NoPifi_d3_rep1

description: Library name: SP000114


title: Cre_NoPifi_d5_rep1

description: Library name: SP000118


title: SA-Y-06-51_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I17


title: SA-Y-01-47_Lu


title: M_40C_bio_rep_2

value: 24 hour culture in REM at 40 degrees Celcius


title: Cre_Pifi48h-at24h_d3_rep1

description: Library name: SP000112

value: Bl6xCast mESC


title: split ePE3 at multiple endogenous sites- Rep 1- I14


title: ERT2CreERT2_NoTamoxifen_d7_rep1

description: Library name: SP000068


title: split ePE3 at multiple endogenous sites- Rep 1- I13


title: M_IFN_bio_rep_2

value: 24 hour culture in REM + 100U/mL IFN g


title: split ePE3 at multiple endogenous sites- Rep 1- I16


title: M_40C_bio_rep_1


title: split ePE3 at multiple endogenous sites- Rep 1- I15


title: M_IFN_bio_rep_1


title: M_Ouab_bio_rep_2

value: 24 hour culture in REM + 70uM Ouabain


title: Cre_NoTamoxifen_d7_rep1

description: Library name: SP000066


title: SA-Y-01-43_Lu

value: male


title: SA-Y-01-46_Lu


title: SA-Y-01_45_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I10


title: M_Ouab_bio_rep_1


title: SA-Y-06-49_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I9


title: CreERT2_Tamoxifen_d7_rep1

description: Library name: SP000064


title: CreERT2_NoTamoxifen_d7_rep1

description: Library name: SP000067


title: SA-Y-01-40_Lu


title: split ePE3 at multiple endogenous sites- Rep 1- I12


title: SA-Y-01-44_Lu


title: M_Kcl_bio_rep_2

value: 24 hour culture in REM + 80mM Kcl


title: CreERT2_NoTamoxifen_d5_rep1

description: Library name: SP000062


title: SA-Y-01-39_Lu

value: lung


title: M_Kcl_bio_rep_1


title: M_Control_bio_rep_3


title: split ePE3 at multiple endogenous sites- Rep 1- I6


title: split ePE3 at multiple endogenous sites- Rep 1- I11


title: M_Control_bio_rep_4


title: ERT2CreERT2_NoTamoxifen_d3_rep1

description: Library name: SP000056


title: ERT2CreERT2_Tamoxifen_d7_rep1

description: Library name: SP000065


title: Met_2_4599

city: London

first: Oscar

last: Maiques

address: Charterhouse Square

department: Tumour Microenvironment

country: United Kingdom

institute: Queen Mary's University of London- Cancer Research UK Barts Centre

data_processing:
Raw data (raw reads) of fastq format were firstly processed through fastp software. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality
Assembly: Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean 2 reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Supplementary files format and content: featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.

source_name: Melanoma lung metastasis

growth_protocol: 4599 murine melanoma cells isolated from different tumour parts (TB, PIF, DIF and MET) were seeded on Fibrillar bovine dermal collagen 1.7 mg/ml (PureCol, Cat. 5005 Advanced BioMatrix) for 24h DMEM+10% FBS.

extract_protocol:
RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) followed by QIAGEN RNeasy Midi Kit. RNA integrity and quality was evaluated using RNA QubitTM assay.
Sequencing was performed using 150bp paired end sequencing strategy using Illumina sequencing platform and PE150 Technology
The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.

treatment_protocol: Media was changed to DMEM+1% FBS during 24h before the RNA extraction.

value: Melanoma lung metastasis

value: Melanoma

tag: tumour part

value: Met