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| Status |
Public on Dec 14, 2022 |
| Title |
SupFig9 maize nCasphi lc gRNA2 rep1 |
| Sample type |
SRA |
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| Source name |
maize mesophyll protoplast
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| Organism |
Zea mays |
| Characteristics |
tissue: mysophyll protoplast
developmental stage: 10-12 days
strain: B73
target locus: Lc
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| Growth protocol |
To grow Arabidopsis plants for protoplast preparation, Col-0 and fwa-4 epi-mutant plants were grown under a 12-hour light/12-hour dark photoperiod and with low light condition for 3-4 weeks. To grow maize plants for protoplast isolation, B73 seeds were soaked in water overnight and planted in half peat moss and half vermiculite. After three days of light incubation, emerged seedlings were transferred to dark until the 2nd leaf was 10-15 cm long. Transgenic T1 plants were screened with half MS plates with 40 μg/ml hygromycin B. For 28 °C treatment of T1 plants, stratified seeds on half MS plates with hygromycin B were incubated at 28 °C and resistant T1 plants were transferred to soil after about a week, and put back to 28 °C for a total of two weeks incubation at 28 °C. T1 plants were then moved to a greenhouse (23 °C) for the rest of the life cycle. To support the growth of albino seedlings in T2 generation, 3% sucrose was supplemented to half MS plates when needed.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Amplicon sequencing: DNA was extracted from protoplast samples and leaves of transgenic plants with Qiagen DNeasy plant mini kit. The amplicon was obtained using two rounds of PCR. Amplification primers for the first round of PCR were designed to have the 3’ sequence of the primers flanking a 200-300 bp fragment of the genomic area targeted by the guide RNA of interest. The 5’ part of the primer contained a sequence which will be bound by common sequencing primers. After 25 cycles of the first round of PCR amplification, the reaction was purified using 1x Ampure XP beads (Beckman Coulter A63881). The eluate was used as template for the second round of PCR using the Phusion enzyme and 12 cycles of amplification. The second round of PCR was designed so that indexes were added to each sample. The samples were then purified using 0.8x Ampure XP beads. Part of the purified libraries were run on a 2% agarose gel to check for size and absence of primer dimer (fragments below 200 bp considered as primer dimer). Then amplicons were sent for sequencing with NovaSeq 6000 or iSeq 100 platform.
Whole genome sequencing: DNA from single Arabidopsis seedlings was extracted with the Qiagen DNeasy plant mini kit and sheared to 300 bp size with a Covaris. Library preparation was performed with Tecan Ovation Ultralow V2 DNA-seq kit. The libraries were sequenced with the NovaSeq 6000 platform.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina iSeq 100 |
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| Data processing |
Amplicon sequencing: Reads were first quality and adaptor trimmed using Trim Galore and then mapped to the target genomic region by the BWA aligner. Sorted and indexed bam files were used as input files for further analysis by the CrispRvariants R package. Each mutation pattern with corresponding read counts was exported by the CrispRvariants R package. After assessing all control samples, a criterion to classify reads as edited reads was established: only reads with a >= 3 bp deletion or insertion (indel, mainly as deletions) of the same pattern (indels of same size starting at the same location) with >= 100 read counts from a sample were counted as edited reads. This criterion is established due to the observation of 1 bp indels and occasionally 2 bp indels with read numbers >100 in control samples. Also, larger indels that occur at very low frequencies (much lower than 100 reads) were observed in control samples. These observations indicate that occasional PCR inaccuracy and low-quality sequencing in a small fraction of reads can result in the indel patterns with corresponding read number ranges as stated above in control samples with the typical sequencing depth in our experiments (1-5 million reads/sample). By employing such stringent criteria, it is believed that the editing signals counted are true signals indicating editing events. Additionally, for FWA gRNA5 and gRNA6 targeted regions, there are long stretches of adenines near these target regions. Due to the high error rate of polymerases amplifying long stretches of adenines, reads with indels only within these stretches of adenines were not counted as true deletions. For amplicon sequencing result analysis of maize protoplast, the criterion to classify reads as edited reads was established: only reads with a >= 3bp deletion or insertion (indel, mainly as deletions) of the same pattern (indels of same size starting at the same location) with >= 10 read counts from a sample were counted as edited reads. The adjustment of the criterion was based on the fact that these amplicon sequencing was of less depth (performed by the iSeq 100) compared to the amplicon libraries of Arabidopsis protoplasts.
Whole genome sequencing for off-target analysis: For variant calling, whole genome sequencing reads were aligned to the TAIR10 reference genome using BWA mem (v0.7.17)53 with default parameters. GATK (4.2.0.0)39 MarkDuplicatesSpark was used to remove PCR duplicate reads. Then GATK HaplotypeCaller was used to call raw variants. Raw SNPs were filtered with QD < 2.0, FS > 60.0, MQ < 40.0, and SOR > 4.0. Raw InDels were filtered with QD < 2.0, FS > 200.0, and SOR > 10.0 and used for base quality score recalibration. The recalibrated bam file was further applied to GATK and Strelka (v2.9.2)40 for SNPs/InDel calling. Only SNPs/InDels called by both GATK and Strelka were used for further analysis. The intersection of GATK and Strelka SNPs/InDels were filtered by removing identical SNPs/InDels in the rdr6-15 background by BedTools (v2.26.0)54. Variants with coverage lower than 30 reads were removed. Variant loci at which the ratio of the reads with the WT allele over the reads with variant allele larger than three were removed. For heterozygous alleles, at a coverage level of 30 reads, the chance of observing WT reads /mutation allele reads >3 is 0.26% (binomial distribution, one-tailed P=0.0026).
Assembly: tair10
Supplementary files format and content: Crispvariant_result, with detected SNV and indels in amplicon sequencing vcf, raw variants called with GATK or strelka2 comparing to tair10 reference genome sequence
Library strategy: amplicon sequencing
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| Submission date |
Jun 23, 2022 |
| Last update date |
Dec 17, 2022 |
| Contact name |
Zhenhui Zhong |
| E-mail(s) |
[email protected]
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| Organization name |
University of California, Los Angeles
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| Department |
Department of Molecular, Cell and Developmental Biology
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| Lab |
Jacobsen Lab
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| Street address |
610 Charles E Young Dr East
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| City |
Los Angeles |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL32388 |
| Series (1) |
| GSE206798 |
Genome editing in plants using the compact editor CasΦ |
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| Relations |
| BioSample |
SAMN29266198 |
| SRA |
SRX15869113 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6263732_SupFig9_maize_nCasphi_lc_gRNA2_rep1_pe_Crispvariant.txt.gz |
332 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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