LA_CMCp_Promastigote_Rep1
To perform -seq, RNA before and after CMC treatment (~800ng) was fragmented, dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), cleaned by Agencourt RNA clean XP beads (Beckman Coulter) and ligated to 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 2ul 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 (20 million reads for each sample).
LA_CMCp_Promastigote_Rep1