title: GSM9264033: Syn, WT, rep 1; Synechococcus elongatus PCC 7942 = FACHB-805; OTHER
library_name: GSM9264033
library_construction_protocol: Cells were harvested in mid-exponential phase at hour 7 in the 12:12 light:dark diel cycle by pelleting 3 x 35-40 mL of cells at OD730 = 0.14-0.22 for 10 minutes (min) at 4,000 rpm at 4C. After pelleting, supernatants were dumped, and the three pellets per sample were flash frozen in liquid nitrogen and stored at -80C until RNA extraction. To extract RNA, cell pellets were thawed on ice in 1 mL phenol stop media (6.5:1 BG-11:phenol stop solution; phenol stop solution is composed of 30:18:1:1 100% ethanol:H2O:phenol:0.5 M EDTA). One pellet from each Mfd_KO biological replicate and two WT pellets (technical replicates) were processed, for a total of 4 samples. Thawed pellets were vortexed briefly and centrifuged at 4,000 rpm for 5 min at 4C, then resuspended in TE buffer. Cells were enzymatically lysed using NEBExpress® T4 lysozyme. Following enzymatic lysis, 2 mL of Buffer RLT (Qiagen) with beta-mercaptoethanol (β-ME) were added and samples were vortexed, followed by addition of 2 mL of 70% ethanol. Samples were vortexed, and the resulting ~4.5 mL lysates were loaded onto RNeasy Midi columns (Qiagen), and RNA was column purified per the manufacturer's instructions. Eluate was further purified by 2x SPRI bead cleanup. To protect RNA, 1 µL of SuperaseIn RNase Inhibitor (Thermo) was added. Samples were then treated with Turbo DNase (Invitrogen) per the manufacturer's protocol to remove contaminating genomic DNA. DNase was heat-inactivated per manufacturer guidelines, and the reactions were purified by 2x SPRI bead cleanup. 19-20 µg total RNA per sample were used to prepare Rend-seq libraries. Library strategy: Rend-seq. rRNA was depleted from 19-20 µg total RNA with MICROBexpress (Invitrogen) following the manufacturer's instructions. Reactions were precipitated in isopropanol. RNA was then fragmented using RNA Fragmentation reagents (Thermo) by first incubating RNA in 10 mM Tris pH 7 at 95C for 2 min, then adding 10x Fragmentation Buffer and incubating at 95C for 25 seconds. The reaction was quenched with Stop Buffer. Samples were precipitated in isopropanol and resuspended in 10 mM Tris pH 7. To size-select RNA, samples were run on a 15% TBE-Urea gel (Thermo) for 65 min at 200 V. 15-45 nt RNA fragments were excised and purified, then precipitated in isopropanol. RNA was dephosphorylated with T4 polynucleotide kinase (NEB) by incubating at 37C for 1 hour, then the reaction was inactivated at 75C for 10 min. RNA was precipitated in isopropanol. Linker-1 (5′ App/CTGTAGGCACCATCAAT/3ddC) was then ligated to the 3′ end of 3 picomoles (pmol) of RNA using T4 RNA Ligase 2, truncated K227Q (NEB) in a reaction containing 25% PEG 8000, T4 RNA Ligase 2 Buffer at 1x, and 100 pmol of Linker-1 by incubating for 2.5 hours at 25C. Ligated RNA was precipitated in isopropanol and resuspended in 10 mM Tris pH 7. The ligated product was then purified by size excision on a 10% TBE-Urea gel (Thermo) run for 50 min at 200 V and precipitated in isopropanol. cDNA was generated by reverse transcription (RT) with SuperScript III (Invitrogen) by first incubating ligated RNA with 25 pmol of RT primer oCJ485 at 65C for 5 min. The remaining reagents (First Strand Buffer at 1x, 20 U SuperaseIn, 5 mM DTT, 0.5 mM dNTPs, and SuperScript III) were then added on ice. The reactions were incubated at 50C for 45 min and then quenched by adding NaOH to a final concentration of 0.1 M and incubating at 95C for 15 min to hydrolyze template RNA. cDNA was purified by size excision on a 10% TBE-Urea gel run for 80 min at 200 V and precipitated in isopropanol. Purified cDNA was resuspended in 10 mM Tris pH 8. Single-stranded cDNA was then circularized with CircLigase (VWR) and additional reagents (2.5 mM MgCl2, 50 µM ATP, CircLigase Buffer at 1x) by incubating at 60C for 1 hour. After an hour, 1 µL of CircLigase was spiked in, and the reaction was incubated at 60C for another hour and then inactivated at 80C for 10 min. The final libraries were amplified by PCR of circularized cDNA and purified by size excision on an 8% TBE gel (Thermo) run for 45 min at 180 V. Amplified libraries were precipitated in isopropanol, resuspended in 10 mM Tris pH 8, and stored at -30C until sequencing.
identifiers.namespace: GEO
identifiers.id: GSM9264033_r1