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Sample GSM7796185

Query DataSets for GSM7796185

Status

Public on Oct 05, 2023

Title

LW13 gradient syringe, below (expt3)

Sample type

SRA

Source name

environmental isolate

Organism

Methylomonas sp. LW13

Characteristics

cell line: environmental isolate
genotype: WT
treatment: below

Treatment protocol

Agarose segments (2 mL) positioned above, at, and below the EPS band were extruded from each syringe through a 23-gauge needle into microcentrifuge tubes.

Growth protocol

For each 10 mL syringe, 1 mL of LW13 cells was mixed with 5 mL NMS1 and 4 mL molten agarose (0.5% w/v, cooled to 55°C). The mixture was poured to the 8 mL marking of a 10 mL plastic Luer-Lok tip syringe fitted with a sterile PTFE filter tip. Solidified syringes were capped with a sterile 20 mm rubber butyl stopper and inlet and outlet needles were pierced through the rubber stopper, and 20 mL of 100% CH4 was flushed through the syringe headspace. Syringes were flushed daily with methane and incubated with the PTFE filter pointing up at 18°C.

Extracted molecule

total RNA

Extraction protocol

Samples were frozen at -80°C until RNA extraction. Thawed aliquots were centrifuged and 600 µL of pre-warmed CTAB extraction buffer was added after removing supernatant. The CTAB buffer consisted of 2% CTAB, 2% polyvinylpyrrolidone 40, 2 M sodium chloride, 100 mM Tris-HCl (pH 8.0), and 20 mM EDTA. Zirconia glass beads were added, and samples were homogenized for 3 minutes at 30 Hz/sec using a bead beater. Samples were extracted twice with 600 µL chloroform-isoamyl alcohol (24:1), and the aqueous phase was mixed with isopropanol and incubated overnight at -20°C. Precipitates were centrifuged for 30 minutes at 16,100 x g at 4°C for 30 minutes and pellets were washed twice with cold 75% ethanol made with DEPC water. Air dried pellets were dissolved in 100 uL DEPC water and treated with DNase I (Ambion) at 37°C for 30 minutes. The DNase I was inactivated by extraction with three volumes of acid phenol:chloroform:IAA (125:24:1, pH 4.5) before pooling RNA from the same segment across all syringes in an overnight precipitation at -20°C in 1 volume isopropanol and 0.8 M LiCl. Precipitates were washed twice with cold 70% ethanol made with DEPC water, air dried, and resuspended in 50 uL DEPC water. Samples were re-purified using RNA Clean & Concentrator-5 (Zymo Research) to remove small RNAs.
RNA libraries for RNA-seq were prepared using NEBNext Ultra II Directional RNA Library Prep with rRNA Depletion kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

counts.csv
analysis.csv

Data processing

Sequence reads were trimmed for adapter sequences/low quality using Trimmomatic v.036 (KBase)
Trimmed sequence reads were mapped to the Methylomonas sp. LW13 genome using HISAT2 v2.1.0 (KBase)
Transcripts were assembled using StringTie v2.1.5 (KBase)
Differential expression matrix was created wusing DESeq2 v1.20.0 (KBase)
Assembly: CP033381.1
Supplementary files format and content: file contains FPKM values of each segment from each independent experiment
Supplementary files format and content: file contains DESeq2 expression matrix from pairwise agarose segment comparisons

Submission date

Sep 22, 2023

Last update date

Oct 05, 2023

Contact name

Aaron W Puri

E-mail(s)

[email protected]

Organization name

University of Utah

Street address

1390 Presidents Circle