Hello everyone ,It 's my first question in biostars. Recently I was annoyed by a question about ssRNA-seq library preparation. there are two question : 1. we know that standard RNA-seq library preparation produce double-strand cDNA.Does two strands of cDNA all be sequenced in sequencing process ? If so, then whether sequencer do the repetitive sequenceing ? I read a sentence from a paper called 《Transcriptom analysis by strand-specific sequencing of complementary DNA》it says：ignoring the orientation of transcription leads to an overestimation of expression level for genes where the level of reverse transcription is comparable with that of forward。Whether it means standard RNA-seq also count antisense transcripts as sense transcripts(if antisense transcripts exiested) ,and that leads to the overestimation of expression level of genes.
2.we know that strand-specific RNA-seq library preparation will get ride of second strand of cDNA to retain the orginal infromation but then cDNA first strand will PCR and synthesize second strand but we know that in sequencing precess ,only the first strand will be sequenced . I wonder whether the special 5' and 3' adapter will not only be used to retain directional information of cDNA first strand ,but also play a role in P5 P7 adapters in sequencing in illumina platform? I refer the A: RNA seq stranded
I hope someone who knows the answer can help me .Thanks advance!