GSM8266837: 429752_N; Homo sapiens; RNA-Seq
Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was extracted from the total RNA using magnetic beads that had poly-T oligos attached to them. The mRNA was then subjected to fragmentation facilitated by divalent cations at an increased temperature within a First Strand Synthesis Reaction Buffer (5X). For the synthesis of the first strand of cDNA, a random hexamer primer and M-MuLV Reverse Transcriptase devoid of RNase H activity were employed. The creation of the second strand of cDNA was followed up using DNA Polymerase I and RNase H. The residual overhangs on the DNA fragments were transformed into blunt ends with the help of exonuclease and polymerase enzymes. Subsequently, the 3' ends of the DNA fragments were adenylated, and adapters with a hairpin loop were attached to prepare the fragments for hybridization. To isolate cDNA fragments that were predominantly 370 to 420 base pairs in length, the AMPure XP system was used to purify the library fragments. PCR amplification was then conducted using Phusion High-Fidelity DNA polymerase in conjunction with Universal PCR primers and an Index (X) Primer. Finally, the PCR products were subjected to purification using the AMPure XP system, and the library's quality was determined with the Agilent Bioanalyzer 2100 system.