Gephyrocapsa huxleyi CCMP1742 transcriptome
Emiliania huxleyi
Further Details
Culture experiments carried out with Gephyrocapsa huxleyi CCMP1742 cells grown in a 3L batches in 5L Nalgene bottles. Cultures were kept in a climate controlled room at 20C, they were continuously stirred and air was constantly provided using an aquarium air pump through a 0.2 um pore size filter (Sartorius, Göttingen, Germany). Light intensity: 105-125 uE. Cell growth was monitored daily using flow cytometry. Cells were harvested at different cell densities by filtering 50 mL of culture over GF/F filters as described above and immediately stored at -80C until further processing. Samples were sequenced individually using Illumina NovaSeq 6000. Library layout: PAIRED. Library design: stranded polyA RNA-seq. Library construction protocol: TruSeq Stranded mRNA polyA library preparations were carried out. Fragments were barcoded using Unique Molecular Identifiers (UMIs) (9 bp length) during library preparation. Identifiers were originally in a separate FastQ file. Then the raw FASTQ file was modified to add the UMI identifiers to the FastQ header. Paired reads from all samples were concatenated and assembled using Trinity (v 2.11.0), with GCC compiler and options --NO_SEQTK and --normalize_by_read_set.
INSDC center name
Royal Netherlands Institute for Sea Research (NIOZ)
Submitter Id
CCMP1742 Transcriptome Trinity
amount or size of sample collected
0.05
collected_by
Ruth Perez Gallego
collection date
2019
culture_collection
National Center for Marine Algae and Microbiota (NCMA)
geographic location (country and/or sea)
missing: lab stock
nucleic acid extraction
RNA was extracted from one quarter of each of glass fiber GF/F filter using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions with the following modifications: after addition of 1 mL lysis buffer supplemented with 10 ul beta-mercaptoethanol to the cut filters, 50ul of Ambion Plant RNA isolation Aid (Ambion, Lithuania) were added to optimize RNA extraction. Filters were bead beated using a mixture of 0.2 mL 0.5 mm diameter and 0.2 mL 0.1 mm diameter glass beads (BioSpec Products, Bartlesville, OK, USA) twice for 30 seconds at a speed of 4 m s-1 with a 30 second dwell using a bead mill homogenizer (Bead Ruptor Elite, Omni International, Kennesaw, GA, USA). After centrifugation of the extracts, 2 ul of SUPERase In RNase Inhibitor 20 U ul-1 (Invitrogen, Carlsbad, CA, USA) was added to the cleared lysate. On-column DNA digestion during RNA extraction was performed using On-Column DNAse I Digestion Set (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer�s instructions. Additionally, RNA eluates were treated twice with TURBO DNA-free kit to remove possible DNA contamination. PCR was performed on RNA extracts to confirm the absence of DNA. 20 U ul-1 (Invitrogen, Carlsbad, CA, USA) was added to the cleared lysate. On-column DNA digestion during RNA extraction was performed using On-Column DNAse I Digestion Set (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer�s instructions. Additionally, RNA eluates were treated twice with TURBO DNA-free kit to remove possible DNA contamination. PCR was performed on RNA extracts to confirm the absence of DNA. The quality and integrity of the RNA extracts was assessed using an Agilent RNA 6000 Nano Kit on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
sample collection method
Filtration over GF/F filter
sample name
CCMP1742 Transcriptome Trinity
sample storage conditions
Frozen
sample storage temperature
-80
scientific_name
Emiliania huxleyi
strain
CCMP1742
water temperature
20