title: GSM3316252: HFSC-RingKO-rep1_RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: FACS-sorted Cells were collected in 0.5mL Eppendorf and processed directly following SMART-seq2 protocol as described in Picelli et al., 2011 with minor modifications. Briefly, 3000 cells per sample were collected and fudge to pellet them, supernatant were removed and cells were directly lysed in  2ul of lysis buffer (0.2% Triton X-100, 4U/uL RNase inhibitor). 1uL of 10uM oligo-dT30Vn and 1uL of 10mM dNTPs were added pipetting well and the samples were incubated 3min ad 72°C. RT step were performed as described in the paper with Superscript III reverse transcriptase enzyme (Invitrogen). Pre-amplification of the obtained cDNA were performed with KAPA HotStart Taq enzyme with High Fidelity buffer. Pre-amplified cDNA were purified with Ampure beads (Agencourt AMPure XP- Beckman Coulter) using Invitrogen Magnet, concentration were assayed with Qubit (Thermo-Fisher Q33216) dsDNA High sensitivity reagents  (Qubit™ dsDNA HS Assay Kit, Thermo-Fisher Q32851)  and quality were verified by Bioanalyzer (Agilent) using Dna High sensitivity kit. 2 nanograms of cDNA were tagmented with100ng of home made Tn5 enzyme and further amplified with KAPA (Kapa HiFi Hotstart KIT). Tagmented amplified DNA were purified with Ampure beads (Agencourt AMPure XP- Beckman Coulter) and the quality of obtained library were controlled at Bioanalyzer with DNA HS assay, loading 1ug of sample, prior sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3316252