From: Charles Plessy <charles.plessy@oist.jp>
Date: Mon, 21 Nov 2022 04:26:26 +0000 (+0900)
Subject: Café
X-Git-Url: https://source.charles.plessy.org/?a=commitdiff_plain;h=b48f09a655de08d1229f1fa072a86ebe1a54c805;p=source%2F.git

Café
---

diff --git a/biblio/36324505.mdwn b/biblio/36324505.mdwn
new file mode 100644
index 00000000..748975e9
--- /dev/null
+++ b/biblio/36324505.mdwn
@@ -0,0 +1,37 @@
+[[!meta title="Improved Nanopore full-length cDNA sequencing by PCR-suppression."]]
+[[!tag Nanopore amplification PCR]]
+
+Bayega A, Oikonomopoulos S, Wang YC, Ragoussis J.
+
+Front Genet. 2022 Oct 17;13:1031355. doi:10.3389/fgene.2022.1031355.
+
+Improved Nanopore full-length cDNA sequencing by PCR-suppression.
+
+[[!pmid 36324505 desc="“We observed [a range of 2.2 to 8.3-fold increase in yield] of amplicons from the Panhandle method compared to the ONT method.”  “Further, the cDNA profile of samples from Panhandle protocol showed a significantly reduced amount of molecules below 600 bp compared to ONT protocol.”   “Reads generated with the ONT protocol showed a marked 3′ bias with only about 40–50% of reads showing full-length coverage of the genes”"]]
+
+
+for each sample total RNA was added together with
+
+RNA +
+1 μL of 10 μm oligo (dT) primer +
+1 μL of 10 mm dNTPs +
+Final volume: 11.6 μL pre-RT reaction.
+
+the reaction was incubated at 72°C 3 min. 4°C 10 min, 25°C 1 min, then held at 4°C.
+
+A 10.4μL reverse transcription (RT) reaction containing
+1 X Maxima H Buffer,
+1 μL RNaseOut (NEB),
+2 μL of 100 μm TSO,
+2 μL of 5M Betaine (Sigma-Aldrich),
+and 1 μL of Maxima H reverse transcriptase was added to the pre-RT reaction and the reaction incubated as shown in Supplementary Protocol.
+
+Following reverse transcription,
+
+5 μL of cDNA was used
+in a 50 μL PCR reaction containing
+1 μL of 10 μm PCR primer and
+25 μL of 2x LongAmp Taq Master mix (NEB).
+
+PCR was performed as shown in Supplementary Protocol. 20 PCR cycles were used. Following PCR, 1 μL of exonuclease (NEB) was added to each reaction and incubated for 15 min at 37°C followed by 15 min at 80°C and the samples were purified using 1x AMPure XP beads (Beckman Coulter).
+
diff --git a/tags/amplification.mdwn b/tags/amplification.mdwn
index 7c8520fe..c2b6bb67 100644
--- a/tags/amplification.mdwn
+++ b/tags/amplification.mdwn
@@ -18,6 +18,11 @@ concentration and annealing temperature) were investigated by [[Dai and coll.,
 complex sample, relatively long ITR (compared to primer length), high
 [annealing temperature], and high concentration of primer should be used.”.
 
+Suppression PCR was used to prepare transcriptome library for Oxford Nanopore
+Technologies sequencers ([[Bayega and coll., 2022|biblo/36324505]]).
+Transcript coverage and amplicon length were improved.  Surprisingly, yield was
+also higher.
+
 ## Other methods
 
  - Primary Template-directed Amplification (PTA), a kind of MDA with terminators