|
| Status |
Public on Nov 09, 2018 |
| Title |
Salt m6A IP Rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
4 week old rosette leaves
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
background ecotype: Col-0
genotype: UBQ10:NTF/ACT2p:BirA
tissue: rosette leaves
age: 4 weeks
|
| Treatment protocol |
Two weeks after sowing, plants were treated with Hoaglands solution with or without 50mM NaCl added. Three days later they were watered again with Hoaglands solution with or without 100mM NaCl. They were watered with or without 100mM NaCl every three days for a total of 3 waterings.
|
| Growth protocol |
Plants were grown in soil under 16 hour light/8 hour dark cycle at 22°C
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Following control or salt treatment, rosette leaves were collected 4 hrs after first light. Leaves were immediately placed on liquid N2 and ground using a mortar and pestle. RNA was extracted using Qiazol (Qiagen) according to manufacturers instructions. 2 rounds of PolyA+ selection was performed using the Dynabeads mRNADirect kit (Thermo). miCLIP-seq was performed using 3 μg of polyA+ selected mRNA per replicate. Samples were placed at 75 degrees Celsius for 5 minutes and snap cooled on ice for 2 minutes. Samples were brought to 686 uxL with nuclease free water. Next, 10 uxL RNaseOUT, 200 uxL 5X IP buffer (250 mM tris HCl, 750 mM NaCl, 0.5% vol/vol Igepal[CA-6300]), and 3 uxL of m6A antibody (Abcam ab151230) were added to samples which were rotated at 4 °C for 2 hours. While rotating, Protein A bead slurry was washed twice with 1mL 1X IP buffer and resuspended in 1 mL 1X IP buffer, which was supplemented with 0.5 mg/mL BSA and rotated for 2 hours at 4 °C. After 2 hours, RNA was transferred into 3 cm cell culture dish and cross linked twice with 0.15 J/cm2 UV light in an agilent stratalinker. Protein A beads were then pelleted using magnetic strand and washed twice using 1 mL 1X IP buffer. 250 uxL of bead mixture and RNA samples were placed into a 2 mL tube and rotated for 2 hours at 4 °C. After 2 hours, beads were pelleted using a magnetic stand, supernatant was removed and stored at -80 °C as unbound supernatant. Bead bound samples were removed from beads by adding 95 uxL proteinase K buffer, 5 uxL of proteinase K, and treated for 45 minutes at 50 °C, agitating every ten minutes. Supernatant was removed and stored at -80 °C as the m6A+ sample. Beads were washed twice using 300 uxL 1X IP buffer. Supernatant from both washes were also stored as m6A+ samples. All samples were kept at -80°C overnight and precipitated using NaOAc. m6A+ samples were then pooled after resuspension in nuclease free water. m6A+ and – were then prepared into libraries using the strand-specific sequencing library preparation as previously described (Silverman et al, 2014).
Strand specific libraries were constructed as previously described (Silverman et al, 2014).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
polyA+ RNA, m6A+ fraction
|
| Data processing |
In order to call m6A peaks reads from all samples were aligned to the Col-0 reference genome/annotation file TAIR10 using STAR (v2.4.0). All non-unique reads were discarded from downstream analysis. Mapped read coverage was split into + and minus strand fractions and macs2 callpeak was then used to call m6A peaks on each strand, respectively, using parameters --nomodel --extsize 50 genome size was set to ½ the transcriptome using -g 32542107 and with an FDR cutoff of < 0.05. m6A peaks are provided in bedgraph format.
Genome_build: TAIR10
Supplementary_files_format_and_content: m6A peaks are presented in bed format with respect to treatment.
|
| |
|
| Submission date |
Jun 27, 2018 |
| Last update date |
Nov 09, 2018 |
| Contact name |
Stephen James Anderson |
| E-mail(s) |
sandersonrbt@gmail.com
|
| Organization name |
University of Pennsylvania
|
| Department |
Biology
|
| Lab |
Brian Gregory
|
| Street address |
433 South University Avenue
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL13222 |
| Series (2) |
| GSE108852 |
N6-methyladenosine-mediated structural changes are associated with transcript cleavage and stability in Arabidopsis |
| GSE116334 |
Profiling of N6-methyladenosine (m6A) in salt-treated and control Arabidopsis |
|
| Relations |
| BioSample |
SAMN09501077 |
| SRA |
SRX4312805 |
