title: NextSeq 500 sequencing; CLIP-Seq experiment of Escherichia coli RelA wild type and RelA::C289Y mutant strains to identify specific RNAs that bind RelA
design: CLIP-Seq experiment of Escherichia coli RelA wild type and RelA::C289Y mutant strains to identify specific RNAs that bind RelA
library_construction_protocol: 300 ml of E. coli ΔrelA cells expressing wild type RelA or RelA:C289Y from a p15A plasmid carrying an arabinose inducible promoter were grown in M9 medium with all supplements to an OD600 of 2.0.The cultures were transferred to a Bio Assay dish and placed in a UV chamber at 800mJ/cm2 to crosslink RNA-protein complexes. The bacterial cells were lysed by vigorous shaking at 30Hz for 10 min and subsequently cooled on ice. The lysed cultures were centrifuged at 16000g, 4°C for 15 min.For each lysate 30µl of anti-RelA antibody (ADAR BIOTECH) was added. Incubated for 1h at 4°C with rotation. This was followed by the addition of the NP-T buffer washed and resuspended protein A sepharose beads (Sigma P6649). The lysate-bead mixture was rotated for another 1h at 4°C.  Each sample was treated with 2.5 units of benzonase nuclease (Sigma E1014) and 1mM MgCl2 present in 100 μL NP-T buffer. Incubated in a thermomixer for 10min at 37°C with 72 g shaking, followed by cooling on ice. This was followed by washing the beads once in 500 μL High-salt buffer and two times with 500 μL CIP buffer (100mM NaCl, 50mM Tris–HCl pH 7.4, 10mM MgCl2). The beads were separated from the supernatant by centrifugation followed by addition of 100 μL CIP solution (10 units of calf intestinal alkaline phosphatase Sigma P4978 in 100 μL CIP buffer). Incubated in a thermomixer for 30 min at 37°C with 72 g shaking, followed by cooling on ice. The beads were washed once with 500 μL High-salt buffer and two times with 500 μL PNK buffer (50mM Tris–HCl pH 7.4, 10mM MgCl2, 0.1Mm spermidine). The beads were subsequently resuspended in 100 μL PNK buffer containing 10 units of T4 polynucleotide kinase (Thermo Fisher Scientific EK0031) and 10 μCi of γ-32P-ATP. Incubated at 37°C for 30 min, then the beads were washed three times in 500 μL NP-T buffer and resuspended in 20 μL Protein loading buffer (60mM Tris–HCl pH 6.8, 0.01% bromophenol blue, 2% SDS, 10% glycerol, 1.5% DTT). Incubated at 95°C for 3 min, centrifuged and supernatant collected.    300 ml of E. coli ΔrelA cells expressing wild type RelA or RelA:C289Y from a p15A plasmid carrying an arabinose inducible promoter were grown in M9 medium with all supplements to an OD600 of 2.0.  The cultures were transferred to a Bio Assay dish and placed in a UV chamber at 800mJ/cm2 to crosslink RNA-protein complexes.  The supernatants were run on 15% SDS-PAGE and subsequently transferred to nitrocellulose membrane. The membrane was subjected to overnight exposure to a phosphor screen. Next day, the membrane region corresponding to RelA-RNA complex was cut out and suspended in 400µl of proteinase K buffer (100mM Tris–HCl pH 7.9, 10mM EDTA, 1% SDS) containing 0.4mg of proteinase K (Sigma P2308) and 10 units of Recombinant RNase inhibitor (RRI Takara 2313A). Incubated at 37°C for 30 min with shaking at 112 g, followed by addition of 100µl of Proteinase K buffer containing 9M urea and incubated under the same conditions. This was followed by centrifugation at 16000g for 2 min and 450 μL of the supernatant was subjected to phenol: chloroform: isoamyl alcohol treatment by subjecting it to incubation for 5min at 30°C in a Thermomixer with shaking at 112 g. Centrifugation at 16000g for 12 min at 4°C and 400µl of the aqueous phase was subjected to precipitation at -80°C for 30 min with 1.2mL ice-cold 99.5% ethanol, 40 μL 3 M NaOAc pH 5.2, and 1 μL GlycoBlue (Thermo Fisher Scientific AM9516). Centrifuged at 16000g for 30min at 4°C and subsequently the pellet was washed with 300µl of ice cold 80% ethanol. Centrifuged again at 16000g for 30min at 4°C, pellet dried and resuspended in 10µl of nuclease free water.   For library preparation, 400ng of RNA was subjected to treatment with FastAP (Thermo Fisher Scientific EF0651), Turbo DNase (Thermo Fisher Scientific AM2238) and RRI. Incubated at 37°C for 30 min, followed by cleaning with Zymo Clean and Concentrator 5 kit (Zymo Research R1013). Then 5µl of RNA was incubated with 1 ul of 100 uM barcoded adapter at 70°C for 2 min, then placed on ice. Then subsequently a ligation mix was added and incubated at 22°C for 2h. All the ligation samples with individual barcode adaptors were pooled together and cleaned using the Zymo Clean and concentrator kit. Following cleaning, ribozero (Illumina 20037135) treatment was done to remove ribosomal RNAs.  Further cleaning of the rRNA depleted RNA was carried out using the KAPA beads (KAPA Biosystems kit KK8000).  Following this, 1µl of 100µM template switching primers bioAR2 and 3Tr3 TSO were added to the RNA sample. Incubated at 72°C for 3 min and at 42°C for 2 min. Then the template switching master mix consisting of SMARTScribe RT enzyme (Takara 639538) was added to the RNA sample with oligos. Incubated at 42°C for 60 min and 70°C for 10 min. This was followed by cleaning with 1.5X KAPA beads and then 1.0X KAPA beads. PCR was done with the cDNA to amplify the library, followed by cleaning with 1.5X KAPA beads to retain fragments ranging from 150-300bp.