From 094fd9794ff62bda03e9c44e3410d6f5e0adf015 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Fri, 14 Mar 2025 16:06:05 +0100
Subject: [PATCH] Athaliana seedlings

---
 .gitattributes                                |   1 +
 assays/BacterialGrowthRates/isa.assay.xlsx    | Bin 10078 -> 10079 bytes
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 14425 bytes
 .../protocols/.gitkeep                        |   0
 .../R401onAthalianaSeedlingsAgarPlates.md     |   3 +++
 7 files changed, 4 insertions(+)
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/dataset/.gitkeep
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep
 create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md

diff --git a/.gitattributes b/.gitattributes
index 50a354a..e09f562 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -12,3 +12,4 @@ assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -t
 assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
 assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Mono-association[[:space:]]experiment[[:space:]]of[[:space:]]R401[[:space:]]on[[:space:]]Athaliana[[:space:]]seedlings[[:space:]]in[[:space:]]agar[[:space:]]plates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
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diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep
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diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md
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+++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md	
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+## Mono-association experiment of R401 on A. thaliana seedlings in agar plates
+
+This protocol is adapted from (25). In brief, *A. thaliana* seeds were sterilized, germinated, and transferred to 1⁄2 MS agar plates without sucrose, as described before. After transfer of seedlings, plants were grown for another 14 days under the same conditions. R401 wild-type and mutants were grown in 50% TSB overnight as described before. Bacterial cells were pelleted by centrifugation, washed 3x in 10 mM MgCl2 and OD~600~ was measured and adjusted to 0.0001. Agar plates were flushed with 15 ml of bacterial suspension for 5 min. The bacterial suspension were removed, and plants were carefully transferred to new 1⁄2 MS agar plates. After 24 h, roots were cut using a sterile scalpel and collected in pre-weighed, sterile 2 ml tubes containing 1 steel bead (3 mm diameter). Tubes were weighed again to assess the root fresh weight. Subsequently, roots were ground in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm at 15 s intervals. Then, 250 μl of sterile 10 mM MgCl2 were added to each tube and roots were ground again under the same conditions. Each sample was subsequently 5x 1:10 diluted in sterile 10 mM MgCl2. Undiluted samples and each dilution were plated on 50% TSA square plates, dried and left to grow at 25 °C until single colonies appeared. Pictures were taken and single colonies were counted blinded.
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