alias: GSM4412592
title: GSM4412592: KO3_A1_P2; Mus musculus; RNA-Seq
library_construction_protocol: RNA isolation and library construction was done using a modified version of the Smart-seq2 protocol as recently described (Sade-Feldman et al, Cell 2018). Briefly, 22µl of Agencourt RNAClean XP SPRI beads (Beckman Coulter, A63987) was added and mixed in the 96 well plates containing sorted single cell lysates. After 10 minutes incubation, plates were placed on a DynaMag-96 side skirted magnet (Invitrogen, 12027) for 5 minutes, followed by supernatant removal and two washes with 75% ethanol. Next, dried beads were mixed with 4µl of mix-1 containing 1µl (10µM) RT primer (IDT, DNA oligo) 5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′; 1µl (10mM) dNTPs (Thermo-Fisher, R0192); 1µl (4U/µl) Recombinant RNase Inhibitor (Clontech, 2313B); and 1µl nuclease free water, and plates were placed in a thermocycler for 3 minutes at 72°C. After RNA denaturation, reverse transcription (RT) step was done by adding, 7µl of mix-2 containing 0.75µl nuclease free water; 2µl 5X Maxima RT buffer (Thermo-Fisher, EP0753); 2µl (5M) betaine (Sigma-Aldrich, B0300-1VL); 0.9µl (100mM) MgCl2 (Sigma-Aldrich, M1028); 1µl (10µM) TSO primer (Qiagen, RNA oligo) 5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′; 0.25µl (40U/µl) Recombinant RNase Inhibitor (Clontech, 2313B); and 0.1µl (200U/µl) of Maxima H Minus Reverse Transcriptase (Thermo-Fisher, EP0753) was added and mixed followed by 90 minutes incubation at 50°C and 5 minutes incubation at 85°C. For cDNA amplification, 14µl of mix-3 containing 1µl nuclease free water; 0.5µl (10µM) ISPCR primer (IDT, DNA oligo) 5′-AAGCAGTGGTATCAACGCAGAGT-3′; and 12.5µl 2X KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2602) was added and mixed, and plates were placed for 3 minutes at 98°C, followed by 21 cycles at [98C for 15 seconds, 67C for 20 seconds and 72C for 6 minutes] with final extension at 72C for 5 minutes. Removal of primer dimers after cDNA amplification was done by adding 20µl of Agencourt AMPureXP SPRI beads (Beckman Coulter, A63881) to each well, incubation for 5minutes, placement of plates on a DynaMag-96 side skirted magnet for 5 minutes, followed by two 75% ethanol washes and resuspension of dried beads with 20µl TE buffer (Tekanova, T0228). This step was repeated to ensure complete removal of primer dimers. Quantification of the concentration of each cell was done on the Cytation-5 plate reader (BioTek) by using the Qubit dsDNA high sensitivity assay (Invitrogen, Q32854). For each plate, representative wells were also evaluated for cDNA size distribution to ensure quality of sorted cells, using the High-sensitivity DNA Bioanalyzer kit (Agilent 5067-4626). The Nextera XT library Prep kit (Illumina, FC-131-1096) was used to generate libraries for next-generation sequencing. After this step barcoded single cells were grouped into a single tube, followed by sequencing on a NextSeq 500 sequencer (Illumina) using the 75 cycles kit, with paired-end 38-base-reads and dual barcoding 8-base-reads.
identifiers.namespace: GEO
identifiers.id: GSM4412592