title: GSM4998728: Bifidobacterium longum W13 transcriptom sequence 2; Bifidobacterium longum; RNA-Seq
library_construction_protocol: Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). Then RNA quality was determined using 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). High-quality RNA sample (OD260/280=1.8~2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0,>10μg) is used to construct sequencing library. RNA-seq strand-specific libraries were prepared following TruSeq RNA sample preparation Kit from Illumina (San Diego, CA), using 5μg of total RNA. Shortly, rRNA removal by RiboZero rRNA removal kit (Epicenter), fragmented using fragmentation buffer. cDNA synthesis, end repair, A-base addition and ligation of the Illumina-indexed adaptors were performed according to Illumina's protocol. Libraries were then size selected for cDNA target fragments of 200-300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, Paired-end libraries were sequenced by Illumina NovaSeq 6000 sequencing (150bp*2.).