GSM5740148: C3b; Toxoplasma gondii; RNA-Seq
GSM5740148
Samples in TRIzol were thawed on ice and all RNA was extracted using a standard chloroform/isopropanol RNA purification, per the manufacturer's instructions. Following the final ethanol wash, pellets were resuspended in 4 μL of Pre-RT buffer (RNase-free water (ThermoFisher 10977023), dNTPs (ThermoFisher R0194; 2.5 mM), and RNA inhibitor (TaKaRa 2313; 10 units/reaction), Oligo-dT (IDT; 2.5 μM)) except for sample C3c, which was resuspended in 8 uL of Pre-RT buffer and divided into two aliquots. For reverse transcription, 6 μL of Reverse Transcriptase Buffer (5x First strand buffer (TaKaRa 639538), Betaine (Sigma B0300; 1 M), DTT - Dithiothreitol  (TaKaRa 639538; 1.66 mM), TSO - Template Switch Olgio (IDT; 1 μM), MgCl2 (ThermoFisher AM9530G; 7 mM), and SmartScribe Reverse Transcriptase (TaKaRa 639538; 100 units/reaction)) were added to each of the samples (28). Per the instructions for the reverse transcriptase kit, the samples were run on a thermocycler with the following steps: 1) 42°C for 90 minutes; 2) 50°C for 2 minutes; 3) 42°C for 2 minutes; 4) repeat steps 2 and 3 (10x); 5) 70°C for 15 minutes. Next, cDNA was amplified using HiFi Hotstart Ready Mix (ThermoFisher 50-196-5217) with ISPCR (IDT; 0.1 μM) and Lambda Exo (New England BioLabs MO262S; 1 units/reaction) for 12 or 14 total cycles. Amplified cDNA was purified using AMPure XP beads (Beckman Coulter A63881) and concentration was determined by Qubit 4.0 using the 1x dsDNA HS kit (ThermoFisher Q33230). Nextera XT indices were used to generate libraries from 1 ng of cDNA material for each sample following the instructions provided by Illumina (Illumina FC-131-1096 and FC-131-2001). Library preparations were purified using AMPure XP beads and checked for quality using the Agilent Bioanalyzer at Stanford Functional Genomics Facility (SFGF). A sequencing pilot was done with libraries pooled in equal amount and run on one NextSeq500 lane with high output, 1x75 sequencing. Using the results from this sequencing a second library was prepared based on the fraction of unique Toxoplasma reads to total reads for a library in an attempt to generate approximately the same number of Toxoplasma reads per sample. The second library was run over two lanes NextSeq500 lanes with high output, 1x75 sequencing at the SFGF. Results from the different lanes for each clone or subclone were pooled prior to alignment.