title: RNAseq of  S. mediterranea: GLORI converted libraries from kiaa1429 (RNAi) animals, having reduced m6A levels, used for detecting m6A sites replicate 3
design: RNA-seq following conversion of non-methylated A by GLORI: For the GLORI protocol, 100-150 ng of double poly-A selected RNA per sample was used. RNA was fragmented to an approximate size of 300 nt using the Invitrogen RNA Fragmentation Reagents kit (Invitrogen). Cleanup with Dynabeads MyOne Silane was performed after each step, except those where the sample was eluted in water. DNase treatment and dephosphorylation were conducted by incubating each sample with T4 Polynucleotide Kinase (NEB), TURBO DNase, and FastAP (Thermo Scientific) for 30 minutes at 37C in 5 FNKBuffer (1:1:2 ratio of T4 PNK buffer:FastAP buffer:H2O). Subsequently, 3 RNA barcode adapter ligation was performed using 100 pmol (Table below) and 36 U of T4 RNA ligase (NEB) for 1.5 hours at room temperature (23C) for each sample. After the 3 adapter ligation, all samples were pooled, with 20% of the pool retained as a control-RNA sample. The remaining 80% of the pooled RNA underwent GLORI treatment as described by Shen et al 26. This involved deamination of the RNA with glyoxal and sodium nitrite, followed by RNA deprotection and purification as outlined in the published protocol 26. cDNA synthesis for both control and treated pools was carried out using the rTd reverse transcription primer (Table below) and SuperScript III Reverse Transcriptase (SuperScript III, 18080051). After synthesis, residual rDT primer was removed by adding 3 L of ExoSAP-IT (Affymetrix, 75001) and incubating at 37C for 12 minutes. RNA hydrolysis was then performed in 1 M NaOH at 70C for 12 minutes. For 5 adapter ligation, 50 pmol of 5iLL-22 DNA adapter (Table below) was ligated with 45 U of T4 RNA Ligase 1 (NEB, M0437M) for 4 hours at room temperature. PCR amplification of cDNA was conducted using the KAPA HiFi PCR Kit (KAPA Biosystems KK2601). The resulting amplified cDNA libraries were then purified using AMPure XP beads (Agencourt, A63881). Sequencing was performed on Illumina sequencing platform.
identifiers.namespace: SUB15093174
identifiers.id: GLORI_kiaa1429_RNAi_3