From 3454c384af211e8a9b47a1038353dc59dc49baf5 Mon Sep 17 00:00:00 2001
From: Vanessa Valencia <vaval103@hhu.de>
Date: Thu, 17 Aug 2023 15:06:56 +0200
Subject: [PATCH] add short protocols
---
assays/rna-seq/isa.assay.xlsx | Bin 2649 -> 2692 bytes
assays/rna-seq/protocols/extract protocol.md | 1 +
.../library construction protocol.md | 1 +
isa.investigation.xlsx | Bin 3212 -> 3259 bytes
.../protocols/growth protocol.md | 1 +
.../protocols/treatment protocol .md | 1 +
6 files changed, 4 insertions(+)
create mode 100644 assays/rna-seq/protocols/extract protocol.md
create mode 100644 assays/rna-seq/protocols/library construction protocol.md
create mode 100644 studies/relative_contribution_of_serine_pathways/protocols/growth protocol.md
create mode 100644 studies/relative_contribution_of_serine_pathways/protocols/treatment protocol .md
diff --git a/assays/rna-seq/isa.assay.xlsx b/assays/rna-seq/isa.assay.xlsx
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diff --git a/assays/rna-seq/protocols/extract protocol.md b/assays/rna-seq/protocols/extract protocol.md
new file mode 100644
index 0000000..d6faefe
--- /dev/null
+++ b/assays/rna-seq/protocols/extract protocol.md
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+Mature leaves were harvested and immediately frozen in liquid nitrogen. RNA was isolated from ground tissue using the Qiagen RNeasy plant mini kit. Residues of DNA were removed with DNase (New England Biolabs). RNA integrity, sequencing library and fragment size were analyzed on a 2100 Bioanalyzer (Agilent). Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina), and quantified with a Qubit 2.0 (Invitrogen).
diff --git a/assays/rna-seq/protocols/library construction protocol.md b/assays/rna-seq/protocols/library construction protocol.md
new file mode 100644
index 0000000..6e15b0d
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+++ b/assays/rna-seq/protocols/library construction protocol.md
@@ -0,0 +1 @@
+Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina). Samples were multiplexed with 12 libraries per lane and sequenced in paired-end mode (150 bp read length) on an Illumina HiSeq 3000 platform.
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diff --git a/studies/relative_contribution_of_serine_pathways/protocols/growth protocol.md b/studies/relative_contribution_of_serine_pathways/protocols/growth protocol.md
new file mode 100644
index 0000000..5011150
--- /dev/null
+++ b/studies/relative_contribution_of_serine_pathways/protocols/growth protocol.md
@@ -0,0 +1 @@
+Arabidopsis thaliana Heynh. Col–0 and its mutant bou‒2 (Li et al., 2003; Rosso et al., 2003), available from the Nottingham Arabidopsis Stock Centre as N370142 and harbouring a T-DNA insertion in the second exon of the BOU gene sequence, were used in this study. For sterilization, Arabidopsis seeds were washed twice with 70% (v/v) ethanol, containing 1% (v/v) Triton X‒100, twice with 100% ethanol and dried. Seeds were grown on 0.5 Murashige and Skoog (MS) medium (Murashige & Skoog 1962) with 0.8% (w/v) agar at pH 5.8. After cold stratification at 4°C to synchronize germination, seedlings were grown at 60% relative humidity, 100 – 150 µmol photons m-2 s-1, 12 h light / 12 h dark, 22°C / 18°C in a 3,000 ppm CO2-enriched atmosphere. Seedlings with two developed leaves were transferred to soil and cultivated under the conditions indicated above.
diff --git a/studies/relative_contribution_of_serine_pathways/protocols/treatment protocol .md b/studies/relative_contribution_of_serine_pathways/protocols/treatment protocol .md
new file mode 100644
index 0000000..26181e2
--- /dev/null
+++ b/studies/relative_contribution_of_serine_pathways/protocols/treatment protocol .md
@@ -0,0 +1 @@
+A. thaliana plants were transferred to ambient CO2 (400 ppm) 24 h prior to harvest. Two sets of plants were harvested (1) ca. 10 min after light onset and (2) 3 h before light onset. The whole rosette of the first set of plants was used for quantification of metabolites and for RNAseq analyses. From the other set of plants, fully mature leaves (number 5 to 7 counted by the order of emergence from the rosette) were cut. Half of these leaves were exposed to [35S]-sulfate for analysis of total sulfate uptake, the flux into the pools of sulfate, proteins, GSH and GLS , as well as GSH and GLS contents. The other half of the leaves was treated in the same way with non-radioactive Hoagland solution and was used for the determination of sulfate and protein contents. Leaf samples were frozen in liquid nitrogen and stored at -80°C until further use.
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