GSM4212312: reporter+EtOH9; Mus musculus; RNA-Seq
Intracellular contents were obtained from live single neurons in M1 of the mouse cortex at P (postnatal day) 30. The collected single-cell contents were expelled into a thin-walled RNA-free PCR tube (Eppendorf, NY), with 1% (final) IGEPAL CA-630 (Sigma-Aldrich, MO), and immediately frozen on dry ice for storage at −80°C until further processing. The cDNA libraries were prepared using the SMARTer Ultra Low Input RNA Kit for Sequencing - v3 (TAKARA Bio) and Nextera XT DNA Library Prep Kit (Illumina) as per the manufacturer's instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies) and Kapa Library Quantification Kit (Kapa Biosystems). The libraries were pooled and diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 (Illumina) and run for 51 cycles using a single-read recipe (TruSeq SBS Kit v3, Illumina) according to the manufacturer's instructions. Illumina CASAVA pipeline (released version 1.8, Illumina) was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter. Additional quality filtering used FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to keep only reads that have at least 80% of bases with a quality score of 20 or more (conducted by fastq_quality_filter function) and reads left with > 10 bases after being trimmed with reads with a quality score of < 20 (conducted by fastq_quality_trimmer function).
GEO Accession
GSM4212312