alias: GSM2120795
title: GSM2120795: P0; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from collected retinas using the TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. DNA-free total RNA was sheared into 200–300 nt fragments by heating (94°C for 3 minutes) with magnesium. Sheared RNA was precipitated by ethanol with GlycoBlue (Ambion) as a carrier. Reverse transcription was carried out using a modified oligo(dT) primer (5'-bio-TTTTTTTTTTTTTTTTdUT TTVN-3', ‘bio’ denotes duo biotin group, ‘dU’ stands for deoxyuricile, ‘V’ represents any nucleotide except T and ‘N’ denotes any nucleotide). Incubate the reverse transcription reaction at 42°C for 2 min before adding Superscript reverse transcriptase II (Invitrogen) to increase specificity. After second strand synthesis, Dynabeads MyOne C1 (Invitrogen) was used to pull down the resulting dsDNA. Incubate the beads with APex Heat-Labile Alkaline Phosphatase (Epicentre) to remove phosphate group, which enables strand specificity at the later PCR step since only the bottom strand cDNA can be ligated and thus amplified. To release dsDNA from MyOne beads, USER enzyme (NEB) was added. The released dsDNA was then end repaired, followed by adding an ‘A’ base at the ends. Illumina paired-end Y linker was ligated and size selected. A 16- cycle PCR was then carried out with Phusion Hot Start High-Fidelity DNA Polymerase (Finnzymes) to generate the final PA-seq libraries.
identifiers.namespace: GEO
identifiers.id: GSM2120795