GSM1465066: RNA-seq high light stress rep2; Synechocystis sp. PCC 6803; RNA-Seq
Harvested Synechocystis cells were centrifuged and resuspended with lysis buffer, 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 5 mM MgCl2, and 1% Triton-X of 300 ul. Further, the resuspended cells were dropped into a mortar packed with liquid nitrogen, and grinded with a pestle. Then the lysate was centrifuged and for efficient RNA extraction from cells, the supernatant was further chemically lysed with QIAzol lysis reagent (Qiagen, Hilden, Germany) of 700 µl for 5 min at room temperature according to miRNeasy mini kit manual (Qiagen). The lysate was incubated with chloroform of 140 µl for 2-3 min at 4 ℃ and centrifuged for 15 min at 3000 rpm. The supernatant was mixed with 1.5 fold 100% ethanol and the RNA transcript was purified through RNeasy mini spin column (Qiagen). To confirm the quality of extracted RNAs, total RNA was visualized using agarose gel electrophoresis. rRNA from total RNA was removed using the RiboZeroTM magnetic kit for bacteria (Epicenter, Madison, WI) according to the manufacturer’s manual. The removal of rRNA was verified using Experion automated electrophoresis system (Biorad, Hercules, CA). The library preparation for transciptome analysis was achieved using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI) according to the manumacturer’s manual. In brief, rRNA-depleted RNA of 50 ng was fragmented and reverse transcribed to cDNA using six random-sequence primers containing a tagging sequence at their 5′ ends. Then cDNA was tagged at 3′ end using the Terminal-Tagging Oligo (TTO). The cDNA tagged at both its 5’- and 3’-end is purified by Agencourt AMPure XP system (BeckmanCoulter, Brea, CA) and amplified by 10-cycle PCR using PCR primers that anneal to the tagging sequences and contain the adaptor sequences required for cluster generation and index code. Excess nucleotides and PCR primers are removed from the library using the AMPure XP System. The quality of DNA library and its concentration was calculated using ExperionTM system and Qubit®2.0 fluorometer (Invitrogen, Carlsbad, CA). Finally, validated DNAs were sequenced using Mi-Seq (Illumina, San Diego, CA) and Miseq® V2 reagent kit of 50 cycles according to manufacturer’s manual.