title: GSM3903962: R128-H-004; Mus musculus; RNA-Seq
library_construction_protocol: Cells were resuspended in RLT Plus buffer (Qiagen) and stored at -80 degrees. RNA extraction was carried out using a RNAeasy Plus micro kit (Qiagen) following the manufacturers instructions. Total RNA quality and quantity was assessed using Agilent 2100 Bioanalyser and a RNA Nano 6000 kit (Agilent Technologies). 30-260ng of Total RNA with a RIN value >8 was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, NEB) Total RNA samples underwent library construction using the NEBNext® Single Cell/ Low input RNA Library Prep Kit for Illumina® (E6420, New England Biolabs®).  One ng of total RNA in 7µL was annealed with cDNA primer to initiate first strand synthesis. Reverse transcription and template switching of the primed samples were followed by amplification of the cDNA by PCR (11 cycles). Amplified cDNA samples were then cleaned using a 1.8x Agencourt AMPure® Beads (Beckman Coulter®). Fragmentation of the cDNA and end repair with 5'phosphorylation and dA-tailing were followed by NEBNext adaptor ligation, U excision, size selection using Agencourt AMPure® XP beads (Beckman Coulter®) and PCR enrichment (8 cycles) of the adaptor-ligated DNA using NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs)(E6440) followed by a clean up of the PCR product using Agencourt AMPure® XP beads (Beckman Coulter®). The libraries were validated using the Agilent 4200 TapStation and kit  (Agilent Technologies) to ascertain the insert size. The Qubit® (Life Technologies) was used to perform the fluorometric quantitation. Following validation, the libraries were normalized to 4nM, pooled together and clustered on the cBot™2 following the manufacturer's recommendations. The pool was then sequenced using a 75-base paired-end (2x75bp PE) dual index read format on the Illumina® HiSeq4000 according to the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3903962