title: GSM8685383: Rattus norvegicus, ratonis epidermis Limbal niche, biol rep2; Rattus norvegicus; RNA-Seq
library_name: GSM8685383
library_construction_protocol: Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and subsequently converted to cDNA using a reverse transcription kit (GeneCopoeia, Rockville, MD, USA). (1) After passing the Total RNA sample detection, eukaryotic mRNA was enriched using Oligo (dT) magnetic beads; (2) Add Fragmentation Buffer for mRNA fragmentation; (3) Using mRNA as a template, synthesize the first and second strands of cDNA and purify the cDNA; (4) Purified double stranded cDNA was subjected to end repair, base A was added, sequencing adapters were added, and fragment screening was performed to recover cDNA of approximately 350bp; (5) Finally, PCR enrichment was performed to obtain a cDNA library. For library preparation, 1 µg of total RNA per sample from rat tissues was subjected to poly(A) selection using oligo(dT) beads, followed by reverse transcription into cDNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) with random hexamer primers. The resulting cDNA was then processed for end repair, phosphorylation, and adenylation according to Illumina's library preparation protocol. Size selection of 300 bp fragments was performed using 2% Low Range Ultra Agarose gel electrophoresis, followed by PCR amplification with Phusion DNA Polymerase (NEB) for 15 cycles. Library quantification was carried out using a TBS380 fluorometer (Turner Biosystems).