GSM6600587: HARD-AP, rep1; Homo sapiens; OTHER
Total RNAs were isolated using the Trizol Reagent. We incubated 75ul HARD-AP beads with  50ug the total RNAs of HEK293 cells for 2 hours.After washing and eluting the samples，we got the HARD-AP samples. The total RNAs were the Input sample. RNA sequencing libraries were prepared from 2μg of input RNAs or HARD-AP purified RNAs with the following modification. We removed ribosomal RNA using Epicentre Ribo-Zero™ rRNA Removal Kit (Human). Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. Using RNA as a template and random oligonucleotides as primers, synthesize the first strand of cDNA, then use RNaseH to degrade the RNA strand, and in the DNA polymerase I system, use dNTP with dUTP instead of dTTP as raw material to synthesize the second strand of cDNA .Purify the double-stranded cDNA, then perform double-end repair and introduce the "A" base at the 3'end and connect the sequencing adapter. At this time, USER enzyme (NEB, USA) was added to degrade the second strand of cDNA containing U. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina)