title: GSM2858911: Day 21_AM_rep2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNAs were extracted by using RNeasy Mini Kit (Qiagen). Purified cDNA was digested with anchoring enzymes (NlaIII), resulting fragments were bound to streptavidin-coated beads (Dynabeads streptavidin M-280), and non-biotinylated cDNA fragments were removed by washing. Adapter-1 (5'– ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGCAGCATG –3', 5'– /Phos/CTGCTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAG TGT/AmMO/ –3') was ligated to cDNA fragments on the beads and after washing digested with EcoP15I. EcoP15I-digested and released fragments (adapter-1- tags) were ligated to adapters-2 (5'– /Phos/AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC/AmMO/ –3', 5'– GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT –3'). Tags sandwiched between two adapters were amplified by PCR using PhusionHigh polymerase and GEX primers (5′- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT CCGATCT -3′ and 5′- CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTC -3′, X; barcodes). The PCR regime consisted of 98°C for 30sec, 10–15 cycles at 98°C for 10sec, 60°C for 30sec and 72°C for 30sec. PCR products were run on an 8% non-denaturing polyacrylamide gel. After staining with SYBR green (Takara Bio), the band at 155–160bp was cut out from the gel, and DNA purified after its elution from the gel pieces.
attributes.tag: GEO Accession
attributes.value: GSM2858911