|
| Status |
Public on Feb 20, 2021 |
| Title |
Con MO input |
| Sample type |
SRA |
| |
|
| Source name |
Animal cap explants from Con MO
|
| Organism |
Xenopus laevis |
| Characteristics |
fraction: Input RNAs
antibody: none (input)
tissue: Animal cap
condition: Control morpholino
|
| Treatment protocol |
Briefly, 600 units of Human chorionic gonadotropin (Daesung Microbiological Labs) were injected into a female Xenopus laevis 12 hours before collecting eggs. Eggs were obtained in 1X MMR (Marc’s Modified Ringer) solution containing 100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES, pH 7.4. Eggs were in vitro fertilized using excised testis. In vitro synthesized mRNAs and/or morpholino oligonucleotides were introduced into the embryos by microinjection using an IM 300 Microinjector (Narishige International USA, Inc).
|
| Growth protocol |
Xenopus laevis were obtained from the Korean Xenopus Resource Center for Research and from Nasco (Wisconsin, USA).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
PolyA+ RNAs were fragmented using fragmentation buffer (Ambion #AM8740) to yield ~200 nt lengths RNA fragments. Polyclonal anti-m6A antibody (Synaptic Systems) was incubated with fragmented RNAs in RIP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP40) supplemented with RNase inhibitors, RNasin (promega) and Superase-in (Ambion), for 4 hours at 4 °C. Along with this, Dynabeads protein A (Invitrogen #10002D) was washed and pre-blocked with 0.5 mg/ml BSA for 2 hours at 4 °C. Beads were then added to antibody-RNA complex and incubated for an additional 1 hour at 4 °C. Bound RNA was eluted after extensive washing with RIP buffer by competitive elution with buffer containing 7 mM m6A (Abcam #ab145715) twice each for 1 hour at 4 °C. Eluted RNAs were then precipitated with ethanol and used for m6A-sequencing library preparation.
Sequencing libraries were prepared using SMARTer Stranded RNA-seq kit (Clontech) according to the manufacturer’s protocol. Purified m6A-seq libraries were sequenced on an Illumina MiSeq platform with 10-20% PhiX control library (Illumina).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Input control for m6A-seq from Con MO
|
| Data processing |
Library strategy: m6A-seq
Illumina Miseq reads from both the input and IP were mapped to the Xenopus laevis v9.2 genome from Xenbase (Karimi, 2018) using Bowtie2 v2.2.6 with the “--local” option (Langmead, 2012).
Aligned m6A-seq reads was first processed using samtools v1.8 (Li, 2009). The read coverage of the aligned m6A-IP reads and RNA input reads were computed for the genes of interest (i.e. known ciliary genes and transcriptional factors) in a strand-specific manner using bedtools v2.26 with command “coverage” and parameters -s and -d (Quinlan, 2010).
Genome_build: Xenopus laevis v9.2
Supplementary_files_format_and_content: .depth files are in BED format and the last column represents the number of m6A-seq reads mapped at the single nucleotide location.
|
| |
|
| Submission date |
Feb 19, 2021 |
| Last update date |
Feb 21, 2021 |
| Contact name |
Young-suk Lee |
| E-mail(s) |
youngl@kaist.ac.kr
|
| Organization name |
KAIST
|
| Department |
Department of Bio and Brain Engineering
|
| Street address |
1113 CMS(E16), 291 Daehak-ro, Yuseong-gu,
|
| City |
Daejeon |
| ZIP/Postal code |
34141 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL22426 |
| Series (1) |
| GSE167139 |
Identification of the FTO-FOXJ1 axis as a conserved regulatory module of embryonic and homeostatic motile ciliogenesis |
|
| Relations |
| BioSample |
SAMN18010365 |
| SRA |
SRX10139790 |
