title: GSM3927454: Ino80E13dKOC; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from freshly micro-dissected embryonic 13.5 cortices using Trizol and RNA Clean & Concentrator Kit. 600ng of total RNA was used for library generation. The ClickSeq procedure used was previously described (Routh et al. 2017). Briefly, total RNA was extracted using Trizol (Invitrogen) according to manufacturer's protocol. The RNA was then treated with DNaseI, cleaned and concentrated using RNA Clean and Concentrator Kit (Zymo Research). Ribosomal RNA was removed using NEBNext rRNA Depletion Kit (NEB). Reverse transcription was carried out with SuperScript II (Invitrogen) following manufacturer's procedures using 1:30 5mM AzdNTP:dNTP and 3'Genomic Adapter-6N RT primer (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN). The reaction was then treated with RNaseH to remove RNA template and cleaned using DNA Clean and Concentrator Kit (Zymo Research). The azido-terminated cDNA was diluted with DMSO and click adaptor (/5Hexynyl/NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT). The click reaction was initiated by adding Cu2+ (Lumiprobe) in presence of vitamin C.  The resulting click ligation was purified using DNA Clean and Concentrator Kit (Zymo). The final amplification step was carried out using 2x One Taq Hot Start Mastermix (NEB), using Illumina universal primer (AATGATACGGCGACCACCGAG) and Illumina indexing primer (CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGT).  The PCR reaction was cleaned using Ampure XP magnetic beads (Beckman) at 1.25x ratio, which size-selects for product size above 200bp. The resulting library was analyzed using TapeStation (Agilent) to check for proper size distribution and quality of DNA.
identifiers.namespace: GEO
identifiers.id: GSM3927454