title: GSM1154020: scnRNAs co-immunoprecipitated with HA-Twi1p-hAGO2Lmut; Tetrahymena thermophila; RNA-Seq
library_construction_protocol: Wild-type Tetrahymena thermophila strains B2086 and CU428 and the complete TWI1 KO strains (Mochizuki and Gorovsky 2004) were used for total RNA preparation. Twi1p was immunoprecipitated (Noto et al. 2010) from either wild-type cells using an anti-Twi1p antibody (Noto et al. 2010) or from the strains expressing HA-Twi1p and HA-Twi1p-hAGO2Lmut (Mochizuki and Kurth in press) using an anti-HA antibody (16B12, Covance). The cDNA libraries were constructed as described (Schoeberl et al. 2012). For multiplex sequencing, oligonucleotides listed below were used. Single-read 36- or 50-base sequences were generated using the Illumina GAII or HiSeq2000, respectively. Sequence reads were filtered as described (Schoeberl et al. 2012). The 3' adapter for multiplex sequencing: 5’-App-NNN NAG ATC GGA AGA GCA CAC GTC T-3ddC; The 5' adapter for multiplex sequencing: 5'-ACA CUC UUU CCC UAC ACG ACG CUC UUC CGA UCU NNN-3'; The reverse-transcription primer for multiplex sequencing: 5’-CAA GCA GAA GAC GGC ATA CGA GAT BBB BBB GTG ACT GGA GTT CAGACG TGT GCT CTT CCG ATC T-3’ (“BBB BBB" = 6 base barcodes for maltiplex sequencing: 5'-CGT GAT-3', 5'-ACT TCG-3', 5'-GCC TAA-3', 5'- TGG TCA-3', 5'- CAC TGT-3', 5'-ATT GGC-3', 5'- GAT CTG-3', 5'- TCA AGT-3', 5'-CTG ATC -3', or 5'-AAG CTA-3'); The PCR amplification primer for multiplex sequencing: 5’-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GAT CT-3’ The RNA was gel-fractionated, and a small RNA population, including scnRNAs (26–32 nt), was used to construct the cDNA library for deep sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1154020