E-MTAB-2588:dek_6-3
Physcomitrium patens
Protocols: Cultures were performed on solid BCD medium overlaid with cellophane. Incubator light conditions were set as long day regime (16 hours light [70 to 80 µmolm-2s-1]/8 hours dark) with a constant temperature of 25C. Wild type and deltadek1 entrained protonemal tissue cultures were collected, homogenized in sterile water and inoculated on BCD medium overlaid with cellophane disc (2 ml of homogenized tissue per plate). Samples for each strain were collected after 6 and 14 days of growth, snap frozen in liquid nitrogen and stored at -80C. The procedure was repeated independently three times. Total RNA was extracted from frozen material using the RNeasy lipid tissue mini kit (Qiagen) with few modifications. Briefly, the frozen tissue was thoroughly homogenized in liquid nitrogen using a mortar and pistil. Approximately 120 mg of powdered tissue was lysed in 1ml QIAzol lysis reagent. Two hundred microliters of chloroform was added and the mixture was centrifuged at 4C. The aqueous phase was collected, 1.5 volume of 100% ethanol was added and the mixture was vortexed. After binding of the RNA to the RNeasy mini spin column, on-column DNaseI treatment was performed to remove genomic DNA. The column was washed with the RPE buffer, dried and RNA eluted in 45µl of RNase-free water. The concentration of RNA was measured and RNA integrity was further assessed using an Agilent 2100 Bioanalyzer (DE54704553, Agilent Technologies, Inc.) with a RNA 6000 LabChip kit. The RNA samples were stored at -80C until sent for sequencing. Library preparation was preformed according to TruSeq RNA Sample Preparation Guide (Illumina, Part # 15008136 REV. A). Succinctly, the total RNA samples were first treated with DNase I to degrade any possible DNA contamination. Then, mRNA was enriched using the oligo(dT) magnetic beads. Finally, mRNA was fragmented into short fragments (about 200 bp). cDNA first strand was synthesized by using random hexamer-primer and cDNA second strand was subsequently synthesized using DNA polymerase I in presence of RNase H. End reparation and 3'-end single nucleotide A (adenine) addition was performed. Finally, sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used to qualify and quantify of the sample library.
ENA checklist
ERC000011
INSDC center alias
German Research Center for Environmental Health
INSDC center name
German Research Center for Environmental Health
INSDC first public
2014-09-04T17:01:11Z
INSDC last update
2014-05-19T11:50:37Z
INSDC status
public
SRA accession
ERS460546
Sample Name
ERS460546
age
6
age
6 day
alias
E-MTAB-2588:dek_6-3
broker name
ArrayExpress
genotype
deltadek1
organism
Physcomitrella patens
organism part
protonemata
title
dek_6-3