title: GSM4143517: Agrp_neurons_RNA_seq_Phf6_cKO-1_Fasted_condition; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed and around 1 mm3 hypothalamic tissue from each mouse was obtained with 1 mm-diatmeter micro punch in ice-cold dissection solution (1xHBSS with 10 mM HEPES and 23 mM D-glucose). Cell dissociation was conducted using Papain Dissociation System kit (Worthington-Biochem: #LK003150), and the cell dissociation procedure was conducted under the guidance of the kit. 1000 tdTomato+ AgRP neurons in 10 ml RNAseq lysis buffer with RNase inhibitor per sample were submitted for cDNA synthesis by using SMART-seq v4 Ultra Low Input RNA kit (Clontech). First-strand cDNA synthesis was primed by the 3' SMART-Seq CDS Primer II A and used the SMART-Seq v4 oligonucleotide for template switching at the 5' end of the transcript. cDNAs were amplified by LD PCR.  PCR II A amplified cDNA from the SMART sequences introduced by 3' SMART-Seq CDS Primer II A and the SMART-Seq v4 oligonucleotide for 15 cycles. PCR-amplified cDNA was purified by immobilization on AMPure XP beads. The beads were then washed with 80% ethonal and cDNA is eluted with elution buffer. Amplified cDNAs were then validated using the Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA kit. 5 ng full-length cDNA output for next-generation sequencing was processed with the Nextera XT DNA library preparation kit (Illumina). Libraries were sequenced on the Illumina HiSeq X Ten.
attributes.tag: GEO Accession
attributes.value: GSM4143517