title: GSM8665888: NTHi_Msup_cell_1, 5 hours growth; Haemophilus influenzae; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using a pre-lysis step, the Maxwell 16 LEV simplyRNA Tissue Kit (Cat# AS1280), and the Maxwell 16 Instrument (Promega) according to the manufacturer's instructions. Briefly, the bacteria pellet was thawed and suspended in 200µL PBS with mutanolysin and proteinase K and incubated for 25 minutes at 37C.  We added 400µL homogenization solution containing 1-thioglycerol, vortexed the sample, put it on ice and added 400µL lysis buffer. Samples were vortexed and transferred to the maxwell LEV cartridge. After extraction, we added an extra DNAse treatment of the RNA with RNase-Free DNase (Qiagen #79254). RNA was purified using the Qiagen RNeasy Mini Elute Clean-up kit according to the manufacturer's instructions. 1µg of purified RNA was sent to the Yale Center for Genome Analysis (YCGA) for sequencing. The Kapa Biosystems rRNA depletion kit was used for ribosomal RNA reduction and samples were sequenced on the Illumina NovaSeq 6000 instrument using a 100bp paired-end read protocol and multiplexed with a target of 10 million reads per sample for bacteria supernatant experiments, 5 million reads for bacteria culture, 75 million reads for H292 cells, and 150M reads per sample for bacteria/cell line culture experiments.
identifiers.namespace: GEO
identifiers.id: GSM8665888_r1