GSM3336648: Smel_FLAG-CoIP2; Sinorhizobium meliloti; RIP-Seq
Cell pellet was resuspended in 5 ml lysis buffer containing 20 mM Tris, pH of 7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, lysozyme, 2 µg/ml Tc and 1 pill protease inhibitor per 40 ml buffer. Cells were lysed by sonication and 40 µl anti-FLAG antibody-bound magnetic beads were added to the cleared lysate. After incubation at 4 °C for 2 h, the beads were collected and washed 3 times with 500 µl lysis buffer containing 2 µg/ml Tc. Purification of CoIP RNA was performed using TRIzol, The total RNA samples were first fragmented using ultrasound (1 pulse of 30 sec at 4°C). First, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase (cycle numbers are indicated in Table 2). The TruSeq barcode sequences which are part of the 5' and 3' TruSeq sequencing adapters are included in Table 2. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 - 500 bp using a preparative agarose gel. An aliquot of the size fractionated pool was analyzed by capillary electrophoresis. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.
GEO Accession
GSM3336648