GSM7593439: k56_ko_2; Mus musculus; RNA-Seq
Metallic beads are added to the mixture and TissueLyzer (Qiagen) was used with the following setting: 50Hz for 2 minutes twice with a fast spin in between.  0.1 ml of chloroform per 0.5 ml of Trizol was added, samples were shaken vigorously for 15 sec and kept for 10 minutes at RT and then centrifuged for 5 minutes at 12,000 x g. The transparent aqueous phase was then transferred to a fresh tube. The RNA was precipitated with 250 l of isopropanol per 0.5 ml of Trizol used for the initial homogenization. Samples were kept on ice for 1 hour and centrifuged for 10 minutes at 4 °C at 12,000 x g. The pellet was washed once with 75 % ethanol by flipping the tube without disturbing the pellet and subsequently centrifuged for 10 minutes at 12,000 x g (4 °C). The ethanol was removed, and the pellet resuspended in 50 l of RNAse and DNAse free water (Ambion). The RNA was quantified with Qbit (Invitrogen) and loaded in a 384 well plate for subsequent cDNA conversion using a previously described protocol 60. Briefly, the RNA undergoes a first-strand conversion in a reaction mixture with SuperScript II reverse transcriptase (Invitrogen), RNase inhibitor (Takara), 5X SuperScript II First-Strand buffer (Invitrogen), DTT (Invitrogen), 5M Betaine (Sigma) MgCl2 (Sigma), and custom locked nucleic acid oligonucleotides. Reverse transcription was performed for 10 cycles followed by inactivation at 70°C for 15 minutes. The total volume of cDNA was added into PCR master mix containing KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and ISPCR primers. The PCR reaction was performed for 22 cycles. Next, PCR products were purified using SeraMag beads (Sigma Aldrich) and 17% PEG, with the final elution in EB buffer (Qiagen). The library quality and size distribution were checked using a High-Sensitivity DNA chip (Agilent Bioanalyzer). For the library preparation, cDNA samples were mixed with 10mM TAPS-NaOH, 5mM MgCl2 pH 8,5, 10% PEG 8K, Tn5 enzyme and water and incubated at 55°C for 6 minutes. After this step the samples were incubated with 0,2% SDS for 5 minutes at RT. Then the samples were pooled after enrichment PCR as described by the manufacturer (illumina) through the use of a dual-index strategy, referred to as index 1 (i7) and index 2 (i5). The sequencing was then performed using a HiSeq 3000 unit at the Single Cell Core Facility (SICOF) at Karolinska Institute (Sweden).