title: GSM8660070: P.aeruginosa PA14, exponential phase, 30 minutes, SHX 100µM biol. Repl. 2; Pseudomonas aeruginosa; RNA-Seq
library_name: GSM8660070
library_construction_protocol: To initiate cell lysis, the thawed samples underwent centrifugation at 8000 revolutions per minute for 5 minutes at room temperature, after which the supernatant was discarded. Following this, 100 microliters of TE buffer containing 800 micrograms per milliliter of lysozyme were introduced to the samples and vigorously vortexed for 30 seconds. The mixture was then allowed to incubate for 10 minutes at room temperature with intermittent brief vortexing. Subsequently, 350 microliters of RLT buffer (from the RNeasy Mini Kit; ID 74106, Qiagen), supplemented with 1% β-mercaptoethanol, were added to the samples. After gently inverting the samples several times, they were incubated at -80°C for 1 hour. The thawed mixture was then loaded into a QIAshredder column (ID 79656, Qiagen) and centrifuged for 2 minutes at 14000 revolutions per minute. Total RNA isolation from the resulting flow-through was performed using the RNeasy Mini Kit (ID 74106, Qiagen) according to the manufacturer's instructions. Any remaining DNA in the extracted RNA samples was eliminated by treatment with the DNA-free DNA Removal kit (ID AM1906, Invitrogen) following the manufacturer's protocol. Library preparation from total RNA followed the methods outlined by Bhattacharyya et al., (2019) and Shishkin et al., (2015). Initially, 1 microgram of total RNA was combined with 10x FastAP buffer (ID EF0651, ThermoFisher Scientific) and subjected to a 90-second incubation at 94°C to induce fragmentation (resulting in fragment sizes of 100 – 300 base pairs). Any residual DNA was eliminated through DNAse treatment using TurboDNAse (ID AM1907, Invitrogen) followed by purification using magnetic beads (RNAClean XP; ID A63987, Beckman Coulter). To ligate 5' phosphorylated barcode oligonucleotides to the dephosphorylated 3' end of RNA fragments, ligation mixes were incubated for 2 minutes at 70°C employing T4 RNA Ligase 1 (ID M0437M, NEB). Subsequently, individual samples were pooled into a single library preparation mixture and purified using the RNA Clean & Concentrator Kit (ID R1017, Zymo Research). Ribosomal RNA was depleted from the pooled samples using RiboZero (ID RZMB12324, Illumina), followed by bead purification as previously described. RNA fragments were transcribed into cDNA using SMARTScribe Reverse Transcriptase (ID 639536, Takara) with incubation at 42°C for 60 minutes followed by 70°C for 10 minutes. After exonuclease treatment (Exonuclease I; ID M0293L, NEB) and bead purification, Illumina index barcode P5 and library-specific primer X01 were introduced during PCR enrichment utilizing AccuPrime Taq DNA Polymerase (ID 12346-086, Invitrogen). Following a final bead purification step, library preparation was concluded. Post-pooling of individual samples and final bead purification, the fragment size profile was assessed using the Fragment Analyzer system (Agilent), and nucleic acid concentration was determined via fluorometric measurement using the Qubit 2.0 system (Thermo Fisher Scientific). Upon successful quality assessment, the final sequencing library underwent paired-end 50 base pair Illumina sequencing at the Genome Analytics Facility (HZI) utilizing a NovaSeq 6000 system.
identifiers.namespace: GEO
identifiers.id: GSM8660070_r1