title: GSM8370070: C7 fungus in Phosphate buffer (a); Penicillium sp. C7(2024); RNA-Seq
library_construction_protocol: Mycelia was harvested on days 6, 9 and 12 by centrifuging at 4000 rpm for 20 min followed by a brief washing with DEPC-treated water (10 mL, 4000 rpm, 20 min). The media from both mono- and co-cultures was filtered through 0.2 µm microplate filter (Millipore) from each set and validated for the growth inhibition activity against pOLYG-mScarlet Mtb H37Rv on respective days. The harvested fungal biomass was ground to fine powder in liquid nitrogen with a mortar-pestle, which was then transferred to 2-3 mL TRIzol Reagent (Invitrogen) to form a slurry and finally thawed. The samples were pipetted several times to ensure complete homogenization and were split into 2-3 RNase-free microcentrifuge tubes (1 mL each) and incubated for 5 min at RT. Chloroform (300 µL) was added to each tube and these were shaken vigorously. The samples were centrifuged at 13,000 x g at 4°C for 15 min. To the upper aqueous layer, an equal volume of 70% ethanol was added to precipitate the RNA, vortexed and the solution was loaded onto spin cartridge columns provided by PureLink RNA isolation kit (Invitrogen). Further purification steps were done according to the manufacturer's guidelines with an on-column DNase I treatment (Zymo Research). The RNA was finally eluted in 40 μL DEPC-treated RNase-free water (Ambion, Invitrogen) and stored at -80°C. RNA concentration and purity were analyzed using Nanodrop spectrophotometer (Thermo Scientific) followed by visual assessments using Bioanalyzer (Agilent 2100) and denaturing formaldehyde agarose gel electrophoresis (1.3% agarose, formaldehyde in 1X MOPS buffer). Total RNA (at least in two biological replicates) from mono- and co-culture conditions were used for mRNA sequencing following ribosomal RNA depletion and cDNA Library prep Libraries for mRNA sequencing were constructed at ZymoResearch from total RNA sample using the Zymo-Seq RiboFree Total RNA Library Prep Kit (Cat # R3000) according to the manufacturer's instructions. Briefly, RNA was reverse transcribed into cDNA, which was followed by ribosomal RNA depletion. After that a partial P7 adapter sequence was ligated at 3' end of cDNAs, followed by second strand synthesis and partial P5 adapter ligation to 5' end of the double stranded DNAs. Lastly, libraries were amplified to incorporate full length adapters under the following conditions: initial denaturation at 95°C for 10 min; 10 - 16 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 60 sec; and final extension at 72°C for 7 min. Successful library construction was confirmed with Agilent's D1000 ScreenTape Assay on TapeStation