alias: GSM1565037
title: GSM1565037: MW-/- SS RNA-seq 2; Mus musculus; RNA-Seq
library_construction_protocol: Adult mouse testes tissue or sorted spermatogenic cells were lysed in Trizol or Trizol-LS reagent and total RNA was isolated according to manufacturer’s recommendations. Small RNA libraries: 20µg of total RNA was size selected on a 15% denaturing polyacrylamide (dPAA) gel to obtain 19-33nt RNA species. The resultant RNA was ligated at its 3’end to the Illumina RA3 using NEB truncated T4 RNA ligase 2 at 20OC for 2 hours. Ligated products were size selected on a 15% dPAA before being ligated at its 5’end to RA5 using Ambion T4 RNA ligase at 20OC for 2 hours. Ligated products were size selected on a 15% dPAA before being reverse transcribed with RTP and superscript III reverse transcriptase. Resultant cDNAs were amplified using RP1 forward primer and indexed reverse primers using NEB Phusion PCR mastermix. Amplified libraries were gel purified on a 2% low-melting agarose gel and sequenced using the Illumina Hi-seq platform. RA3: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/ (ddC: dideoxyC, rN: ribonucleotide, p: phosphate). RA5: rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC. RTP: GCCTTGGCACCCGAGAATTCCA. RP1: AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA. RPI: CAAGCAGAAGACGGCATACGAGATIIIIIIGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA (IIIIII: 6-mer Illumina index sequence). Global 5'RACE and Scriptseq: Total RNA from sorted pachytene spermatocytes, secondary spermatocytes or round spermatids was treated with RQ1 DNase at 37oC for 30 minutes, depleted of ribosomal RNA using the Ribo-Zero kit, and used to generate global 5’RACE libraries as described 7. Post-amplification libraries were size selected on a BluePippin 2% agarose chip using a cutoff of 180-350bp. Ribo-depleted RNA was also used for strand-specific RNA libraries, which was constructed using the Scriptseq v2 RNA-Seq library preparation kit.
identifiers.namespace: GEO
identifiers.id: GSM1565037
attributes.tag: GEO Accession
attributes.value: GSM1565037