GSM9006152: F12, plusCHO, rep2; Plasmodium falciparum; RNA-Seq
RNA was harvested from  tyghtly synchronized cultures at the mid trophozoite stage (33–38 hpi, unless stated otherwise) at ~2 % parasitemia using 20 pellet volumes of TRIrizol (Life Technologies, 15596018). RNA of ap2-hs KO was collected at 37-42 hpi to account for their slower IDC progression (Tintó-Font, E., et al., 2021; PMID:34400833). RNA extraction and treatment with DNase was performed as previously described [Casas-Vila, N., Pickford, A., et al. 2021. PMID: 34313989]. rRNA depletion of 5 μg of RNA was performed using the Rneasy Pure mRNA Bead Kit (Qiagen, 180244) following manufacturers' instructions, and eluted in 18 μl of nuclease-free water. rRNA-depleted RNA was fragmented to ~100-400 bp using the NEBNext Magnesium RNA Fragmentation Module (NEB, E6150S), by adding 2 μl of fragmentation buffer, incubating for 4 min at 94 ºC, placing on ice and immediately adding 2 μl of stop solution. Fragmented RNA was cleaned up using the RNeasy MinElute CleanUp Kit (Qiagen, 74204) and eluted in 11 μl of nuclease-free water. For the first-strand cDNA synthesis, the SuperScript III Reverse Transcriptase kit (Invitrogen, 18080093) was used. Each sample was pre-incubated for 10 min at 70 ºC with 1.25 μl of AT-biased random primers (IDT) and 1 μl of actinomycin (Gibco, 11805017) and then for 2 min at 37 ºC with 8 μl of mastermix containing 1x first-strand buffer, 25 mM DTT, 1.25 mM dNTP (NEB, N0447S) and 1 μl RNasin Plus Ribonuclease inhibitor (40 U/ μl) (Promega, N2611).Next, 1 μl of Superscript III (200 U/ μl) was added to each sample, and incubated for 10 min at 37 ºC, followed by 1 h at 42 ºC. Second-strand cDNA synthesis was achieved by incubating the product of the previous reaction for 2 h at 16 ºC with 55 μl of a mastermix containing 1x second-strand buffer (Invitrogen, 10812014), 0.27 mM dNTP custom mix (10 mM dACG/dU) (Invitrogen, 10297018; Thermo Scientific, R0133), 0.1 μl Ambion RNase H (10 U/μl) (Invitrogen, AM2292), 2 μl DNA Polymerase I (10 U/μl) (Promega, M205A) and 0.5 μl E. coli DNA ligase (10 U/μl) (NEB, M0205S). The final cleanup was performed using the MinElute PCR purification kit (Qiagen, 28006), eluting in 10 μl nuclease-free water. The resultant cDNA was quantified by Qubit HS before library preparation. Library preparation was performed using 5 ng of DNA, as previously described (Llorà-Batlle, O., 2020. PMID: 32577509). NEBNext multiplex oligonucleotides for Illumina (NEB, E7335, E7500 and E7710) were used, and 9 cycles of amplification were performed with the Kapa HiFi HotStart PCR kit (Roche, 7958889001). All purification steps were carried out using AMPure XP beads (Beckman Coulter, 082A63881). Quality and concentration of all libraries was determined using a Tapestation 4200 (Agilent). We obtained ~8-19 million 150 bp paired-end reads per sample using a NextSeq500 System (Illumina).