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| author | Bonface | 2024-02-09 09:41:28 -0600 |
|---|---|---|
| committer | Munyoki Kilyungi | 2024-08-09 13:30:43 +0300 |
| commit | d029d5d7f8ead1f1de8d318045004a4a6f68f5fb (patch) | |
| tree | 33c7ff40e3f953d030ed08f468f7afb1dfcba9e6 /general/datasets/B30_K_1206_Rn/experiment-design.rtf | |
| parent | 769ff7825f5d8d36d541e90534c07f1985899973 (diff) | |
| download | gn-docs-d029d5d7f8ead1f1de8d318045004a4a6f68f5fb.tar.gz | |
Update dataset RTF Files.
Diffstat (limited to 'general/datasets/B30_K_1206_Rn/experiment-design.rtf')
| -rw-r--r-- | general/datasets/B30_K_1206_Rn/experiment-design.rtf | 62 |
1 files changed, 62 insertions, 0 deletions
diff --git a/general/datasets/B30_K_1206_Rn/experiment-design.rtf b/general/datasets/B30_K_1206_Rn/experiment-design.rtf new file mode 100644 index 0000000..e743086 --- /dev/null +++ b/general/datasets/B30_K_1206_Rn/experiment-design.rtf @@ -0,0 +1,62 @@ +<blockquote> +<p><strong>RNA Sample Processing:</strong></p> + +<p>Trizol RNA isolation and RNeasy clean up protocol for whole plants (embryo-derived tissue dissected from 4 days old germinating grains) and the seedling leaves (12 days after planting).</p> + +<p><br /> +☠Grind tissue (9 embryos) with a mortar and pestle in liquid nitrogen<br /> +☠Add 5 ml TRIzol (pre-heated to 60oC) to all samples, vortex until all the tissue is thawed, place in the 60oC waterbath..<br /> +☠Incubate samples at 60oC for 10 minutes, vortexing three times.<br /> +☠Centrifuge @ 4000 x rpm @ 4C for 30 minutes (in Eppendorf 5810R).<br /> +☠While centrifuging, label new set of 15 ml tubes<br /> +☠Transfer supernatant to 15 ml centrifuge tube<br /> +☠Add 1 ml of chloroform. Vortex the sample until color shade is uniform at least 5<br /> +seconds, and incubate at room temperature for 5 minutes.<br /> +☠Centrifuge @ 4000 x rpm for 30 minutes @ 4oC.<br /> +☠While centrifuging, label new 15 ml tubes<br /> +☠Collect the upper aqueous layer (there will be about 3 mls) and transfer to a new 15 ml tube.<br /> +☠Add 0.6 volumes (2 ml) of isopropanol, mix gently, incubate at room temperature for 20 minutes.<br /> +☠Centrifuge @ 4000 rpm for 30 minutes @ 4oC.<br /> +☠Wash the pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at<br /> +4000 rpm for 15 minutes @ 4oC.<br /> +☠Discard supernatant, centrifuge for 5 min, remove the rest of the ethanol<br /> +☠Air-dry the pellet for 10 minutes, inverted on a kimwipe.<br /> +☠Dissolve pellet in 400 ul of DEPC-treated H2O. Resuspend by pipeting up & down a<br /> +few times.<br /> +☠Add 2 ul SuperaseIn. Incubate at 60oC for 10 minutes to resuspend.<br /> +☠Set water bath to 37oC.<br /> +☠Add 50 ul 10X DnaseI Buffer, 45 ul H2O and 5 ul of DnaseI, incubate at 37oC for 1 hr.<br /> +☠Prepare Buffer RLT (Rneasy Clean-up Midi Kit) by adding b-mercaptoethanol (10ul/1ml RLT).<br /> +☠Add 2.0 ml Buffer RLT to the RNA prep and mix thoroughly.<br /> +☠Add 1.4 ml ethanol (96-100%) to the diluted RNA. Mix thoroughly.<br /> +☠Label 15 ml tubes from the kit and place midi columns in them<br /> +☠Apply sample to a Midi column, close tube gently and centrifuge for 20 min at 3000 rpm.<br /> +☠Discard the flow-through.<br /> +☠Add 2.5 ml Buffer RPE to the RNA easy column, close the centrifuge tube gently,<br /> +incubate for 3 min<br /> +☠Centrifuge for 10 min at 3000 rpm. Discard the flow-through.<br /> +☠Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube<br /> +gently, incubate for 3 min<br /> +☠Centrifuge for 10 min at 3000 rpm, remove flow-through<br /> +☠Centrifuge again for another 5 min.<br /> +☠Label new 15 ml tubes from the kit.<br /> +☠Transfer the RNA easy column to a new tube and pipet 250 ul volume of<br /> +RNase-free water directly onto the RNeasy silica-membrane incubate for 1 min<br /> +☠Centrifuge for 5 min at 3000 rpm.<br /> +☠To the same tube add again 250 ul H2O, incubate for 1 min.<br /> +☠Centrifuge for 5 min at 3000 rpm.<br /> +☠Label two sets of 1.5 ml Eppendorf tubes.<br /> +☠Transfer 490 ul to the one tube and 10 ul to another one. Use 10 ul tube for the RNA</p> + +<p>Detailed descriptions of these procedures can be found under the ArrayExpress (http://www.ebi.ac.uk/aerep/?) protocol P-MEXP-4631 (Caldo et al. 2004).</p> + +<p><strong>Replication and Sample Balance:</strong></p> + +<p>3 independent replicates of both parental cultivars Steptoe and Morex were generated for both tissues, embryo and seedling leaf.</p> + +<p><strong>Experimental Design and Batch Structure:</strong></p> +</blockquote> + +<blockquote> +<p>The following are ArrayExpress (http://www.ebi.ac.uk/aerep/?) experiment IDs: E-TABM-111 (leaf, 41 chips) and E-TABM-112 (embryo derived, 156 chips).</p> +</blockquote> |