GSM7872529: N07A-2b; Gallus gallus; miRNA-Seq
All samples were predigested at 56◦C for 1 hour, after which the buffer was changed and then a second 12- hour digest was performed. Digests from methods B and C were centrifuged at 6000 ×G for 1 minute, after which 500 μl supernatant was mixed 1:8 with a binding buffer. DNA bound to the columns was washed with 800 μl buffer PE (Qiagen), then eluted using 2 washes in 17 μl buffer EB (Qiagen)—each with an incubation for 5 minutes at 37◦C. Prior to library construction, small aliquots of each extract were analysed on an Agilent 2200 TapeStation HS chip (Agilent Technologies, Palo Alto, CA, USA) for fragment size estimation and molar concentration. Library blanks and index PCR blanks were also included to evaluate the potential contaminations during the library building process. Adapters were first diluted to 500 μM with ×1 TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, SigmaAldrich). Subsequently, an equimolar concentration of each pair of long and short adapters was mixed together and hybridized through incubation at 95◦C for 1 minute, followed by a decrease in temperature with 0.1◦C/s from 95◦C to 12◦C. After hybridization, adapters AD1 and AD2 were mixed and diluted at a concentration of 10 μM prior to their use in the library construction. First, the libraries were qPCR-quantified using the CommonprimerBGI forward primer and 1 of the indexed reverse primers. Second, subsequent index PCR amplifications used 8 to 15 cycles with CommonprimerBGI forward primer and the indexed reverse primers. Third, because several of the BGISEQ-500 libraries exhibited residual adapter dimers after the initial purification post-index PCR, each purified BGISEQ-500 library was split to 2 aliquots, and 1 of each aliquot was subject to an extra purification to remove any residual primer dimers. All amplified libraries were subsequently sent to BGI for circularization and sequencing on the BGISEQ-500 platform. For circularization, PCR products with different barcodes were pooled together at equimolar concentration to yield a final amount of 80 ng.