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| Status |
Public on Nov 21, 2023 |
| Title |
XR-seq C. elegans xpc-1 CPD 24h |
| Sample type |
SRA |
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| Source name |
L1 larvae
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: L1 larvae
genotype: xpc-1
treatment: UV irradiation induced CPD
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| Treatment protocol |
To obtain L1 larvae, eggs were collected from adult animals by hypochlorite treatment, and kept in M9 buffer at 22℃ for 16 hours with gentle rotation. L1 larvae were exposed to 4,000 J/m2 of UVB radiation (313 nm). The animals were collected in M9 buffer at 5 minutes, 1 hour, 8 hours, 16 hours, 24 hours, and 48 hours after irradiation, and washed until the supernatant became clear. The pelleted C. elegans (~50 μl for each) were then incubated for 2 hours at 62°C with 450 μl of Worm Hirt Lysis Buffer (0.15M Tris pH 8.5, 0.1M NaCl, 5mM EDTA, 1% SDS) and 20 μl of Proteinase K (NEB, cat. no. P8107S). Subsequently, 120 μl of 5M NaCl was added, and the mixture was inverted to ensure proper mixing, followed by an overnight incubation and one hour centrifugation at 4°C.
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| Growth protocol |
The C. elegans wild-type (N2 ancestral) and xpc-1 (tm3886) strains were obtained from the Caenorhabditis Genetics Center and were cultured under standard conditions at room temperature on nematode growth media plates with E. coli strain OP50.
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| Extracted molecule |
other |
| Extraction protocol |
For XR-seq, supernatants were incubated with 5μL RNase A and then 5μL Proteinase K, purified, and then immunoprecipitated with either anti-CPD or anti-(6-4)PP antibodies. For RNA-seq, L1 stage wild-type (WT) and xpc-1 C. elegans were collected in M9 and washed until the supernatant was clear, followed by incubation with TRizol and chloroform. After centrifugation at 14,000g for 15min at 4°C, the aqueous phase was mixed with an equal volume of isopropanol. Following centrifugation, the RNA pellet was washed several times and then resuspended in RNase-free water.
For XR-seq, immunoprecipitations were ligated to the adaptors, purified with the antibody used in the first purification, and DNA damage was reversed by either CPD or (6-4)PP photolyase. After PCR amplification, the library was sequenced. For RNA-seq, quality control, followed by stranded and poly(A) enriched library preparation and sequencing, was performed by Novogene.
Single-end for XR-seq, paired-end for poly(A) RNA-seq.
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
For XR-seq, cutadapt was used to trim reads with adaptor sequence TGGAATTCTCGGGTGCCAAGGAACTCCAGTNNNNNNACGATCTCGTATGCCGTCTTCTGCTTG at the 3′-end and to discard untrimmed reads. Bowtie 2 was used for read alignment to the ce11 reference genome, followed by filtering, sorting, deduplication, and indexing. Post-alignment filtering steps were adopted using Rsamtools. We only keep reads that: (i) have mapping quality greater than 20; (ii) are from chromosome I, II, III, IV, V, and X; and (iii) are of length 19-24 bp. For plotting strand-based average repair profiles of the genes, we selected 7061 genes longer than 1 kilobase pair, situated at least 500 base pairs away from neighboring genes. Each gene was evenly divided into 100 bins from the Transcription Start Site (TSS) to the Transcription End Site (TES), and 25 bins (2 kbp) upstream of TSS, 25 bins (2 kbp) downstream of TES. Bed files of the reads were intersected to the 150 bin-divided-gene list by Bedtools intersect with the following commands -d -wa -F 0.5 -S or -s for TS and NTS, respectively22. We present the descriptive properties of our data in Supplementary Table 1. For RNA-seq, reads were aligned using STAR, followed by a filtering step to remove unmapped reads, reads with unmapped mates, reads that do not pass quality controls, reads that are unpaired, and reads that are not properly paired. We only kept the first read from the mate pair to ensure independent measures. Read counts for each gene were obtained using FeatureCounts.
Assembly: ce11
Supplementary files format and content: bigwig
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| Submission date |
Oct 12, 2023 |
| Last update date |
Nov 21, 2023 |
| Contact name |
Yuchao Jiang |
| E-mail(s) |
yuchaojiang@tamu.edu
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| Organization name |
Texas A&M University
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| Street address |
3143 TAMU
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| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843 |
| Country |
USA |
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| Platform ID |
GPL32326 |
| Series (1) |
| GSE245181 |
Whole-genome analysis of transcription-coupled repair reveals novel transcription events in Caenorhabditis elegans |
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| Relations |
| BioSample |
SAMN37795642 |
| SRA |
SRX22075848 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7838108_XR_xpc_CPD_24h_minus.bw |
1.8 Mb |
(ftp)(http) |
BW |
| GSM7838108_XR_xpc_CPD_24h_plus.bw |
1.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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