From 41cdbbe6c4d4f65d78cda7879416346047bcb661 Mon Sep 17 00:00:00 2001
From: Mekhrona Mirzoeva <memir101@hhu.de>
Date: Thu, 20 Mar 2025 15:43:26 +0100
Subject: [PATCH] 16S rRNA prep protocol, assay
---
.gitattributes | 1 +
assays/DNA Isolation/README.md | 1 +
.../README.md | 0
.../dataset/.gitkeep | 0
.../isa.assay.xlsx | Bin 0 -> 9837 bytes
.../protocols/.gitkeep | 0
.../LibraryPreparation16SrRNAProfiling.md | 3 +++
assays/MicrobiotaReconstitutionFlowpot/README.md | 1 +
.../README.md | 1 +
9 files changed, 7 insertions(+)
create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/README.md
create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep
create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx
create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep
create mode 100644 assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md
diff --git a/.gitattributes b/.gitattributes
index eac0854..4b96006 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -14,3 +14,4 @@ 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
assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
+assays/Library[[:space:]]preparation[[:space:]]for[[:space:]]bacterial[[:space:]]16S[[:space:]]rRNA[[:space:]]gene[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/DNA Isolation/README.md b/assays/DNA Isolation/README.md
index e69de29..0519ecb 100644
--- a/assays/DNA Isolation/README.md
+++ b/assays/DNA Isolation/README.md
@@ -0,0 +1 @@
+
\ No newline at end of file
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/README.md b/assays/Library preparation for bacterial 16S rRNA gene profiling/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep b/assays/Library preparation for bacterial 16S rRNA gene profiling/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx b/assays/Library preparation for bacterial 16S rRNA gene profiling/isa.assay.xlsx
new file mode 100644
index 0000000000000000000000000000000000000000..f69b3ac68bbf290a0cdce64453a6acd50284f8da
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diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md
new file mode 100644
index 0000000..17be9b0
--- /dev/null
+++ b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md
@@ -0,0 +1,3 @@
+## Library preparation for bacterial 16S rRNA gene profiling
+
+The v5v7 variable regions of the bacterial *16S* rRNA gene were amplified in 96-wells plates through PCR using 799F and 1192R primers (PCR I; **Table S4**). PCR I was performed with 0.4 μl DFS-Taq polymerase (BIORON) in 25-μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM 799F, 0.75 μl 10 μM 1192R, 1 μl of isolated DNA and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 25 cycles of 94°C for 30 s, 55°C for 30 s,72°C for 1 min, then a final extension at 72 °C for 10 min. Remaining primers and dNTPs were digested by Antarctic phosphatase and Exonuclease I. To each 25 μl PCR reaction, 1 μl of Antarctic phosphatase (New-England BioLabs), 1 μl of Exonuclease I (New-England BioLabs) and 3 μl of Antarctic phosphatase buffer (New-England BioLabs) were added; the resulting mixture was incubated at 37 °C for 30 min followed by heat inactivation of the enzymes at 85 °C for 15 min. The digested PCR I product was centrifuged at 3,000 rpm and 4 °C for 10 minutes. Then, 3 μl of the supernatant were used as a template in a second PCR, using a unique combination of uniquely barcoded 799F- and 1192-based primers containing Illumina adaptors for each sample (PCR II; **Table S4**). PCR II was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions containing 2.5 μl 10x incomplete buffer, 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM unique forward primer, 0.75 μl 10 μM unique reverse primer, 3 μl cleaned PCR I-product and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 10 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, then a final extension at 72 °C for 10 min. Subsequently, 5 μl of the PCR product were combined with 1 ml Orange DNA Loading Dye, loaded on a 1% agarose gel containing 0.05% EtBr, and run at 110 mV. The expected PCR-product was an approx. 500 bp band containing the variable v5v7 regions, barcodes, and Illumina adaptors. After verification of successful amplification, samples were purified by AMPure XP (Beckman-Coulter) according to the manufacturer’s protocol. The DNA concentrations of each sample were quantified using the Quant-iT dsDNA Assay-Kit (Invitrogen) and pooled per full factorial replicate in equimolar values. All pools were purified twice using AMPure XP and fluorescently quantified using the Quant-iT dsDNA Assay-Kit. All pooled samples were combined into a single pool based on equimolarity and subsequently purified three times using AMPure XP and fluorescently quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). The final concentration was set to 10 ng/μl. Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers (**Table S4**).
diff --git a/assays/MicrobiotaReconstitutionFlowpot/README.md b/assays/MicrobiotaReconstitutionFlowpot/README.md
index e69de29..0519ecb 100644
--- a/assays/MicrobiotaReconstitutionFlowpot/README.md
+++ b/assays/MicrobiotaReconstitutionFlowpot/README.md
@@ -0,0 +1 @@
+
\ No newline at end of file
diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
index e69de29..0519ecb 100644
--- a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
+++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md
@@ -0,0 +1 @@
+
\ No newline at end of file
--
GitLab