|
| Status |
Public on Mar 22, 2022 |
| Title |
sgRNA [WIXL122] |
| Sample type |
RNA |
| |
|
| Source name |
Xenopus, sgRNA control
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: Tadpoles
treatment: sgRNA
mecp2 status: wild type
|
| Treatment protocol |
Cas9 target sites were selected using CHOPCHOP on the X. laevis J-strain 9.2 version, which facilitated the selection of guide RNA with no predicted off-targets. We used the default settings to generate a list of target sequences. We selected 4 targets from this list for each of the L and S forms. This selection was based primarily on the ranking provided by CHOPCHOP and as a function of their location on MeCP2 to target exons coding for the methyl-CpG-binding domain (MBD, exons 2 and 3) and Transcriptional repression domain (TRD, exon 3). The sgRNA were resuspended to 100 μM in 0.1X Tris EDTA (pH 8.0). Then, an equimolar sgRNA mix was made using 2 μl of each sgRNA, for a total of 16 ul. The Cas9 RNP complex was formed by mixing 75 pmol of the sgRNA mix with 75 pmol of Cas9 in annealing buffer (5 mM HEPES, 50 mM KCl, pH 7.5), for a total volume of 100 μL, then incubated at 37C for 10 minutes. The RNP was kept frozen at -20C until the day of the injection. To generate MeCP2 knockdown models of Rett syndrome, Xenopus embryos were fertilized and maintained at 14C until the 4-cell stage. Each cell was injected with ~2 nL RNP per injection resulting in a final amount of 1.5 fmol of RNP per injection per cell.
|
| Growth protocol |
Xenopus embryos and tadpoles were housed at 18C with a 12/12 h light/dark cycle in 0.1X Marc’s Modified Ringer’s (MMR) medium. Tadpoles were fed 3x/week with sera Micron Nature fry food.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tadpoles were sacrificed at stage 50 of development and lysed using a QIAGEN TissueLyser II bead mill (30 Hz, 2 x 30 sec) in a 2 mL tube with a 2.4 mm steel beads (Omni) and 1 mL phosphate buffered saline buffer at 4oC. 250 mL of lysate was used for RNA extraction using a QIAGEN RNeasy mini kit. RNA samples were DNAse treated.
|
| Label |
biotin
|
| Label protocol |
Microarray measurements were performed using the GeneChip Xenopus laevis Genome 2.0 Array (Affymetrix; Santa Clara, CA) at the Advanced Biomedical Laboratories (Cinnaminson, NJ).
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| |
|
| Hybridization protocol |
RNA samples were processed using a Nugen Ovation PICO WTA System V2 kit. The resulting cDNAs were purified using a Qiagen MinElute PCR Purification Kit following the modifications outlined in the Nugen protocol. The cDNAs were fragmented and labeled using a Nugen Encore Biotin Module. Hybridization solutions were prepared by combining the fragmented, biotin-labeled cDNAs with hybridization cocktail (Affymetrix Hybridization, Wash, and Stain Kit). The mixtures were incubated in a thermal cycler at 99 °C for 2 min followed by 45 °C for 5 min. then loaded on Xenopus laevis Genome 2.0 arrays and incubated for 16-20 hours at 45 ºC and 60 rpm in an Affymetrix Hybridization Oven 645.
|
| Scan protocol |
Following hybridization, arrays were washed and stained on Affymetrix Fluidics Station 450s using the Affymetrix FS450_0001 protocol with the stains and buffers supplied in the Affymetrix Hybridization, Wash, and Stain Kit. The stained arrays were scanned at 532 nm using an Affymetrix GeneChip Scanner 3000.
|
| Description |
Experiment 1: CRISPR edited model of Rett syndrome
|
| Data processing |
Microarray data were extracted from CEL files and Robust Multi-array Average (RMA) normalized in Matlab (Mathworks; Natick, MA) and output with linear scaling.
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| |
|
| Submission date |
Mar 21, 2022 |
| Last update date |
Mar 22, 2022 |
| Contact name |
Richard Novak |
| Organization name |
Harvard University
|
| Department |
Wyss Institute
|
| Street address |
3 Blackfan Cir
|
| City |
Boston |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL10756 |
| Series (1) |
| GSE199049 |
Target-agnostic discovery of Rett Syndrome therapeutics by coupling computational network analysis and CRISPR-enabled in vivo disease modeling |
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