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Sample GSM8247371

Query DataSets for GSM8247371

Status

Public on May 03, 2024

Title

cox11 150, ZT6, rep1

Sample type

SRA

Source name

Protonema and young gametophores

Organism

Physcomitrium patens

Characteristics

tissue: Protonema and young gametophores
plant line: 150
genotype: COX11 KO
zeitgeber time: 6

Growth protocol

Protonema grown for one week on solid PpNH4 medium under continuous illumination was disrupted using a T 25 UltraTurrax (IKA) and used for the inoculation of a 100 mL Erlenmeyer flask with 20 mL of liquid PpNH4. After one week of growth under continuous illumination, the plant was harvested, disrupted and used for the inoculation of a 250 mL Erlenmeyer flask with 50 mL of liquid PpNO3. After one week of growth under continuous illumination, the plant was harvested, disrupted and used for the inoculation of a 500 mL Erlenmeyer flask with 100 mL of liquid PpNO3. After one week of growth, the plant material was distributed into a layer of miracloth arranged on top of a plastic cylinder, inside of a Magenta™ box filled with fresh liquid PpNO3. The plant was in contact with the medium through the filter, allowing growth under this “hydroponics” system. Plants in closed Magenta™ boxes were grown for 15 days under a short day regime (light:dark 12h:12h). The day of harvesting, plants were immediately snap frozen at the corresponding zeitgeber time (ZT0, ZT2, ZT6). For plants harvested at ZT0, Magenta™ boxes were enclosed in aluminium foil at the beginning of the night period (ZT12) and samples were snap frozen at ZT0 in a dark room illuminated with dim, green light. For plants harvested at ZT2 or ZT6, plants were snap frozen directly in the growth chamber, avoiding shading the plant until it had been frozen. Frozen samples were kept at -80 ºC until used.

Extracted molecule

polyA RNA

Extraction protocol

Frozen samples were powdered using a cold mortar and pestle, in presence of liquid nitrogen. Approximately 150 mg of powder were used for RNA extraction using the RNeasy Plant Mini Kit, ref. 74904 (Qiagen).
RNA was used to build cDNA libraries using the kit QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen), which exploits oligo-d(T) primers therefore enriching the cDNA library in cDNA representative of mRNA, but not ribosomal RNA or other types of RNA. Libraries were then sequenced with a depth of 5 million of reads using a NextSeq 500 System (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

A14

Data processing

The reference genome (Physcomitrium patens v3.3) from the JGI Plant Gene Atlas (Lang et al., 2018) was indexed using Bowtie 2 (v2.2.5) (Langmead & Salzberg, 2012). Reads were then aligned using Bowtie 2 and bam files were generated with SAMtools (v 1.6) (Danecek et al., 2021). HTSeq-count (v 2.0.2) (Putri et al., 2022) was used to align the mapped reads to the genes provided in the annotation file (Ppatens_318_v3.3.gene.gff).
Assembly: Physcomitrium patens v3.3 from the JGI Plant Gene Atlas
Supplementary files format and content: csv files contain counts per each sample

Submission date

May 02, 2024

Last update date

Jul 17, 2024

Contact name

Tomas Morosinotto

E-mail(s)

[email protected]

Organization name

University of Padova

Department

Department of Biology