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authorBonface2024-02-09 09:41:28 -0600
committerMunyoki Kilyungi2024-08-09 13:30:43 +0300
commitd029d5d7f8ead1f1de8d318045004a4a6f68f5fb (patch)
tree33c7ff40e3f953d030ed08f468f7afb1dfcba9e6 /general/datasets/UTHSC_BXD_Young_Adult_Eye_RNAseq_TPM_Log/experiment-design.rtf
parent769ff7825f5d8d36d541e90534c07f1985899973 (diff)
downloadgn-docs-d029d5d7f8ead1f1de8d318045004a4a6f68f5fb.tar.gz
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+<p>Total RNA was extracted using Trizol<sup>&reg;</sup>&nbsp;reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer&rsquo;s instructions. Approximately 30 mg of PFC tissue was added into a 2 ml tube containing 700ul QIAzol Lysis Reagent and one 5 mm stainless steel bead (Qiagen, Hilden, Germany). The tissue was homogenized for 2 minutes in a Tissue Lyser II (Qiagen, Hilden, Germany) with a speed frequency of 30 r followed by incubating for 5 minutes.140&nbsp;<a name="_Hlk32821176">&micro;</a>l chloroform was added into the homogenate, shaken vigorously for 15 seconds, and centrifuged for 15 minutes at 12,000 x&nbsp;<em>g</em>&nbsp;at 4&nbsp;℃. 280 &micro;l upper aqueous was then transferred into a new collection tube containing 500 &micro;l 100% ethanol. The mixture was loaded into a RNeasy mini‐spin column (Qiagen, Valencia, CA, USA), once with Buffer RWT and twice with Buffer RPE purification. All RNA had been treated with DNase to avoid DNA contamination, and verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA with OD260/280 &gt; 1.8 and RIN &gt; 8.0 were used for library preparation</p>