LM-Cas9_Polysome_PolyA_Rep2
To enrich for poly (A) RNA, total RNA was incubated with Dynabeads mRNA DIRECT (Invitrogen) as per manufacturers instruction and poly (A) selected RNA was extracted. The poly (A) RNA was fragmented and dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter) and ligated to a 3 linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor (NEB). The ligated RNA was cleaned from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme (Agilent). The RNA was subsequently degraded using 2 ul of 1 M NaOH and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to a 3 adapter using high concentration T4 RNA Ligase 1 (NEB) and cleaned of excess adapter by using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter ligated cDNA was PCR enriched using NEBNext High-Fidelity (NEB) polymerase (9 PCR cycles) and sequenced in a Nextseq system (Illumina) in paired end mode (40 million reads for each sample).
LM-Cas9_Polysome_PolyA_Rep2