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Sample GSM8586640

Query DataSets for GSM8586640

Status

Public on Dec 20, 2024

Title

WT, rep1

Sample type

SRA

Source name

Nostoc sp. PCC 7120

Organism

Nostoc sp. PCC 7120 = FACHB-418

Characteristics

cell type: Nostoc sp. PCC 7120
genotype: WT
chip antibody: Anti-FLAG M2 (Sigma, M8823)
treatment: UV-crosslinked sample

Treatment protocol

WT and cells of Alr1700-3xFLAG strain were irradiated with UV light (1.6 mJ/cm2) to crosslink the proteins to their interacting RNA. After harvesting, cells were resuspended in NT-P buffer (50 mM NaH2PO4, 300 mM NaCl, 0.05% Tween, pH 8.0) supplemented with protease Inhibitor (cOmplete EDTA-free, Roche). Cells were mechanically lysed in a pre-chilled Precellys homogenizer (Bertin Technologies) using 9 cycles of 10 s shaking at 6,000 rpm followed by a pause of 5 s. Glass beads were removed by centrifugation at 1,500 x g for 2 min. Next, membranes were pelleted by centrifugation at 21,000 x g for 30 min at 4 ºC. 2 mL of clarified lysate was incubated with 80 µL of anti-FLAG M2 magnetic beads (Sigma) prewashed 5 times in NT-P buffer. From this point on, all the washing steps were performed with 2 mL of buffer. The samples were incubated at 4 °C while gently rotating for 1 h. Beads were washed three times with high-salt NT-P buffer (50 mM NaH2PO4, 1 M NaCl, 0.05% Tween20, pH 8.0) and twice with cold NT-P buffer. Elution of the RNA-protein complexes were carried out by resuspending the beads in 50 µL of TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) supplemented with 0.5 µg/µL FLAG peptide (Sigma) and 2 µL Ribolock RNase inhibitor (life Technologies) at 4 °C for 15 min, while gently shaking at 900 rpm.

Growth protocol

Nostoc sp. PCC 7120 WT and Alr1700-3xFLAG strain were grown photoautotrophically with constant shaking under standard conditions (30 ºC and 50 µE illumination) in liquid BG11 medium. Filaments were collected by centrifugation at 3,270 x g for 5 min at room temperature, washed and then resuspended in BG110 (medium without a combined nitrogen source) to induce heterocyst differentiation for 24h.

Extracted molecule

total RNA

Extraction protocol

Proteins from the eluted RNA-protein complexes were digested in 400 µL proteinase K buffer (50 mM Tris-HCL, pH 6, 5 mM EDTA and 0.5%SDS) with 400 µg of proteinase K for 30 min at 37 °C. Another 400 µL of proteinase K buffer + 9M urea was added for an additional incubation of 30 min at 37 °C. RNA fragments were isolated using phenol-chloroform and chloroform extractions. RNA was later treated with 2U of TURBO DNAse (Life Technologies) to eliminate DNA traces, followed by RNA cleanup using RNA Clean & Concentrator columns (Zymo Research).
Synthesis of cDNA was facilitated by SMARTScribe reverse transcriptase (TaKaRa), which adds the loop adapter at the 3’-end in the first-strand synthesis step and the SMART oligo at the 5’-end in the subsequent template switching and extension step. Annealing of the loop adapter to RNA was achieved by incubating both at 72°C for 3 min and 42°C for 2 min. All components were added to this mixture according to the manufacturer's instructions, including the SMART oligo, 100 U SMARTScribe reverse transcriptase (TaKaRa), and 40 U of Ribolock RNase inhibitor (Life Technologies), and the reaction was incubated at 42°C for 90 min and 70°C for 10 min, followed by cooling to 12°C. After cDNA synthesis, the samples were excised from 3% Nusieve agarose gels and purified with the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), using NTC buffer to solubilize the gel slices. The cDNA was amplified in 22 PCR cycles with Phusion high-fidelity DNA polymerase (New England BioLabs) using Illumina 8-nt index primers for both sides, and PCR samples were purified using the Agencourt AMPure XP system (Beckman Coulter) according to the manufacturer’s instructions. All individual amplified cDNA libraries were pooled in equimolar ratios and size selected between 100 - 320 bp using the Pippin Prep system (Sage System) according to the manufacturer’s instructions and subsequently purified using the Agencourt AMPure XP system (Beckman Coulter).

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 2000

Description

CLIP-seq

Data processing

Quality control was performed using FASTQC
Reads were aligned to Nostoc genome using bowtie2 (alignment in mode --very-sensitive-local)
Peak calling was carried out by PEAKachu (https://github.com/tbischler/PEAKachu)
Assembly: ASM970v1
Supplementary files format and content: A table for the peak calling output is provided (peak_table.xlsx). Columns include the replicon, position and strand of the detected peaks. In addition, for each peak it is also shown the log2foldchange of the comparison between the targetome of PatR-3xFLAG and the background (WT), adjusted p-value and the number of reads counted in each peak per biological replicate.

Submission date

Oct 22, 2024

Last update date

Dec 20, 2024

Contact name

Manuel Brenes-Álvarez

E-mail(s)

[email protected]

Organization name

University of Freiburg

Department

Genetics and Experimental Bioinformatics