GSM8773109: P15 Purkinje cells target library rep 2; Mus musculus; RNA-Seq
For miRNA and target libraries, RNAs were isolated along with Spy3-Ago2 using HaloTag-SpyCatcher3 conjugated to a HaloLink resin from the sample. The miRNA/target and input samples (whole lysate) were run on a SDS-PAGE gel, the gel was cut between 70 kDa and 150 kDa. RNAs were then extracted from the gel using phenol/chloroform and purified with the RNA Clean & Concentrator-5 kit. For input libraries, RNAs were purified from lysates using MyONE Silane beads RNAs were ligated to RNA adapters on both ends (5' RNA adapter 5biotin/rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrNrNrNrNrNrNrNrNrNrN and 3' RNA adapter /5Phos/r(N:25252525)r(N:25252525)r(N:25252525)rUrGrGrArArUrUrCrUrCrGrGrGrUrGrCrCrArArGrG/3SpC3). After reverse transcription with a DNA primer DP3 (CCTTGGCACCCGAGAATTCCA), the library was amplified with DNA primers DP3 and DP5 (GTTCAGAGTTCTACAGTCCGACGATC). To construct the libraries for high-throughput sequencing, DNA primers TS Primer-X ( CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA, N=standard Illumina barcodes) and TS Primer-1 (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC) containing Illumina barcodes, sequencing primer binding sites and Illumina TruSeq indexes for multiplexing were introduced using PCR. After quantification and quality control, libraries were pooled and run on a NextSeq 2000 in a 100 bp single end run.